Immunogenic composition comprising a fusion peptide derived from superantigen toxoids

ABSTRACT

The present disclosure provides immunogenic compositions useful in prevention and treatment of Staphylococcus aureus infection. In particular, the disclosure provides multivalent oligopeptides, fusion proteins comprising two or more staphylococcal superantigen (SAg) proteins, or any fragments, variants, or derivatives thereof fused together as a single polypeptide in any order.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.16/633,664, filed Jan. 24, 2020, which is a U.S. National Phaseapplication of PCT/US2018/043687, filed on Jul. 25, 2018, which claimsthe benefit of U.S. Provisional Patent Application No. 62/537,706, filedJul. 27, 2017, all of which are incorporated herein by reference intheir entirety.

This application contains subject matter related to U.S. patentapplication Ser. No. 14/899,993, filed Dec. 18, 2015, now U.S. Pat. No.9,815,872, which is incorporated by reference herein in its entirety.

GOVERNMENT RIGHTS

This invention was made with Government support under AI111205 awardedby the National Institutes of Health. The Government has certain rightsin the invention.

INCORPORATION OF SEQUENCE LISTING

A sequence listing containing the file named “IBV_193252_SubstituteSeqList_ST25.txt”, which is 35,037 bytes (measured in MS-Windows®),contains 15 sequences, and was created on Apr. 20, 2021, is providedherewith and is incorporated herein by reference in its entirety.

BACKGROUND

Staphylococcus aureus (SA) is a gram-positive human pathogen that causesa wide range of infections from skin and soft tissue infections (SSTI)to life threatening sepsis and pneumonia. It is a leading cause ofhospital- and community-associated infections worldwide (Brown et al.,2009, Journal/Clin Microbiol Infect, 15(2):156-164). The range ofpathologies reflects the diverse abilities of SA to escape the immuneresponse using a plethora of virulence factors: the superantigenic andpore-forming toxins, coagulase, capsular polysaccharide, adhesins,proteases, complement inactivating exoproteins, and other innateresponse modifiers (Powers and Wardenburg, 2014, Journal/PLOS Pathogens,10(2):e1003871).

Since its first emergence in the 1960s methicillin-resistant SA (MRSA)has become endemic in healthcare settings worldwide (Diep, et al., 2006,J Infect Dis, 193 (11):1495-1503). Since the 1990s, community associatedMRSA strains (CA-MRSA) emerged, and are posing a major global challenge(Bassetti, et al., 2009, Int J Antimicrob Agents, 34 Suppl 1:S15-19;Bradley, 2005, Semin Respir Crit Care Med, 26 (6):643-649; Chambers,2005, N. Engl J Med, 352 (14):1485-1487.). There have hence beenincreasing efforts directed towards the development of vaccines andtherapeutics for S. aureus infections.

Alpha hemolysin (a-toxin, ma) is a major virulence factor in SApneumonia and SSTI (Bubeck Wardenburg and Schneewind, 2008, J Exp Med,205 (2):287-294; Kennedy, et al., 2010, J Infect Dis, 202(7):1050-1058). Recently, cytolytic short peptides known as phenolsoluble modulins (PSMs) were identified as key virulence factors thatlyse neutrophils, the main line of defense against S. aureus (Wang, etal., 2007, Nat Med, 13 (12):1510-1514). Another related cytolytic shortpeptide of staphylococci is known as delta hemolysin or delta toxin(Stoxin) the key marker of S. aureus quorum sensing system (agr)(Novick, et al., 1993, EMBO J, 12 (10):3967-3975). A recentepidemiological study in a cohort of patients with SA bacteremia showsinverse correlation between probability of sepsis and pre-existingantibodies to ma, PSM-α3, as well as δ-toxin (Adhikari, et al., 2012, JInfect Dis, 206 (6):915-923).

Superantigens (SAgs) constitute a large family of pyrogenic toxinscomposed of staphylococcal enterotoxins (SEs) and toxic shock syndrometoxin 1 (TSST-1). In contrast to conventional antigens that undergoproteolytic processing by antigen presenting cells and are presented asMHC/peptide complex to T cells, SAgs cross link T cell receptor (TCR)with MHC Class II and activate up to 30% of T cells (Schlievert, 1993,Journal/The Journal of Infectious Diseases, 167(5):997-1002) leading tomassive release of cytokines and chemokines, enhanced expression as wellas activation of cell-adhesion molecules, increased T-cellproliferation, and eventually T-cell apoptosis/anergy. This sequence ofevents can culminate in Toxic Shock Syndrome (TSS), a life-threateningcondition characterized by rash, hypotension, fever, and multisystemdysfunction (Bohach et al., 1990, Journal/Crit Rev Microbiol,17(4):251-272). Antibodies play an important role in protection againstTSS, thus individuals that do not seroconvert towards the offendingtoxin due to hypo responsive T-cells (Mahlknecht et al., 1996,Journal/Hum Immunol, 45(1):42-45) and/or T-cell dependent B-cellapoptosis (Hofer et al., 1996, Journal/Proc Natl Acad Sci USA,93(11):5425-5430) are more likely to experience recurring bouts.Furthermore, at lower non-TSS inducing concentrations SAgs impact thevirulence of S. aureus strains through induction of a local excessiveinflammatory response.

A major challenge in development of multivalent S. aureus vaccinesincluding superantigens is that there are more than 20 different SAgsand there is a wide range of variability in SAg presence in clinicalisolates because most SAgs are on mobile genetic elements, such asplasmids or pathogenicity islands (Staphylococcal enterotoxin K (SEK),Staphylococcal enterotoxin Q (SEQ)), lysogenic phages (Staphylococcalenterotoxin A (SEA)), or antibiotic resistance cassettes, like SCC mecStaphylococcal enterotoxin H (SEH) (Omoe et al., 2002, Journal/J ClinMicrobiol, 40(3):857-862). Based on an extensive literature reviewencompassing over 6000 clinical isolates, the most widely representedsuper antigens (SAgs) appear to be toxic shock syndrome toxin 1 (TSST-1)and Staphylococcal enterotoxin C (SEC), followed by SEA, Staphylococcalenterotoxin D (SED), and Staphylococcal enterotoxin B (SEB). More recentstudies show the emergence of SEK and SEQ, primarily due to circulationof the USA300 clone (Proft and Fraser, 2003, Journal/Clinical andExperimental Immunology, 133(3):299-306). Monoclonal antibodies andvaccination against multiple SAgs have been found to partially protectagainst SA sepsis in mice. Significant protection has been reportedagainst pneumonia in rabbits using multivalent immunization with variouscombinations of detoxified SAgs and cytolysins (Spaulding et al., 2012,Vaccine 30(34):5099-109; Salgado-Pabon et al., 2014, J Infec Dis, 210(5):784-792).

SUMMARY

In one aspect, this disclosure provides for an attenuated Staphylococcusaureus-derived superantigen (SAg) SEA toxoid or an immunogenically orantigenically active fragment, variant, or derivative thereof,comprising four mutations relative to wild-type SEA, the four mutationscorresponding to the L48R, D70R, Y92A, and H225A mutations in SEQ ID NO:4. In certain aspects, the toxoid or fragment, variant, or derivativesthereof, has decreased superantigenic activity and/or is less virulentthan a SEA toxoid comprising SEQ ID NO: 3, while maintainingimmunogenicity. In certain aspects, the attenuated SEA toxoid orfragment, variant, or derivative thereof comprises an amino acidsequence that is at least 90% identical to SEQ ID NO: 4. In certainaspects, the attenuated SEA toxoid or fragment, variant, or derivativethereof comprises SEQ ID NO: 4. And, in certain aspects, the attenuatedSEA toxoid or fragment, variant, or derivative thereof has less than50%, less than 40%, less than 30%, less than 20%, less than 10%, lessthan 5%, less than 3%, less than 2%, or less than 1% of thesuperantigenic activity of a SEA toxoid comprising SEQ ID NO: 3. It willbe understood that the nomenclature used herein to describe pointmutations (e.g. “L48R”) are in comparison to wild-type SAg proteinswhich do not contain the N-terminal Methionine that was required forheterologous expression.

In another aspect, the disclosure further provides for a multivalentoligopeptide comprising a fusion of two or more attenuatedStaphylococcus aureus-derived superantigen (SAg) toxoids orimmunogenically or antigenically active fragments, variants, orderivatives thereof as described elsewhere herein arranged in any order,wherein the SAg toxoids or fragments, variants, or derivatives thereofcan be the same or different, and wherein at least one of the SAgtoxoids is a SEA toxoid described elsewhere herein. In certain aspects,the oligopeptide comprises a fusion of three or more SAg toxoids orfragments, variants, or derivatives thereof. In certain aspects, theoligopeptide has decreased superantigenic activity and/or is lessvirulent than a SAg fusion protein comprising SEQ ID NO: 5. In certainaspects, the oligopeptide maintains the immunogenicity of the SAg fusionprotein comprising SEQ ID NO: 5. In certain aspects, the oligopeptidehas less than 50%, less than 40%, less than 30%, less than 20%, lessthan 10%, less than 5%, less than 3%, less than 2%, or less than 1% ofthe superantigenic activity of a SAg fusion protein comprising SEQ IDNO: 5. And, in certain aspects, the oligopeptide is completelyattenuated.

In certain aspects, the multivalent oligopeptide comprises one or moreof a staphylococcal toxic shock syndrome toxin-1 (TSST-1) attenuatedtoxoid; a staphylococcal enterotoxin B (SEB) attenuated toxoid; or anycombination thereof. In certain aspects, the TSST-1 attenuated toxoidcomprises three mutations relative to wild-type TSST-1, the threemutations corresponding to the L30R, D27A, and I46A mutations in SEQ IDNO: 1 and an amino acid sequence at least 90% identical to SEQ ID NO: 1.In certain aspects, the SEB attenuated toxoid comprises three mutationsrelative to wild-type SEB, the three mutations corresponding to theL45R, Y89A, and Y94A mutations in SEQ ID NO: 2 and an amino acidsequence at least 90% identical to SEQ ID NO: 2. In certain aspects, theSEA attenuated toxoid comprises four mutations relative to wild-typeSEA, the four mutations corresponding to the L48R, D70R, Y92A, and H225Amutations in SEQ ID NO: 4 and an amino acid sequence at least 90%identical to SEQ ID NO: 4. In certain aspects, the TSST-1 toxoidcomprises the amino acid sequence SEQ ID NO: 1. In certain aspects, theSEB toxoid comprises the amino acid sequence SEQ ID NO: 2. In certainaspects, the SEA attenuated toxoid comprises the amino acid sequence SEQID NO: 4. In certain aspects, the multivalent oligopeptide comprises theamino acid sequence SEQ ID NO: 6.

In certain aspects, at least two SAg toxoids or fragments, variants, orderivatives thereof described elsewhere herein are each associated via alinker. In certain aspects, the linker comprises at least one, but nomore than 50 amino acids selected from the group consisting of glycine,serine, alanine, and a combination thereof. In certain aspects, thelinker comprises (GGGS)_(n) (SEQ ID NO: 13) or (GGGGS)_(n) (SEQ ID NO:14), wherein n is a integer from 1 to 10. In certain aspects, the linkercomprises (GGGGS)_(n) (SEQ ID NO: 14). In certain aspects, n is 3 (SEQID NO: 15).

The multivalent oligopeptide can further comprise a heterologouspolypeptide. In certain aspects, the heterologous polypeptide comprisesa His-tag, a ubiquitin tag, a NusA tag, a chitin binding domain, aB-tag, a HSB-tag, green fluorescent protein (GFP), a calmodulin bindingprotein (CBP), a galactose-binding protein, a maltose binding protein(MBP), cellulose binding domains (CBD's), anavidin/streptavidin/Strep-tag, trpE, chloramphenicol acetyltransferase,lacZ (β-Galactosidase), a FLAG™ peptide, an S-tag, a T7-tag, a fragmentof any of the heterologous polypeptides, or a combination of two or moreof the heterologous polypeptides. In certain aspects, the heterologouspolypeptide comprises an immunogen, a T-cell epitope, a B-cell epitope,a fragment thereof, or a combination thereof.

The multivalent oligopeptide can also further comprise an immunogeniccarbohydrate. In certain aspects, the immunogenic carbohydrate is asaccharide. In certain aspects, the immunogenic carbohydrate is acapsular polysaccharide or a surface polysaccharide. In certain aspects,the immunogenic carbohydrate is selected from the group consisting ofcapsular polysaccharide (CP) serotype 5 (CP5), CP8,poly-N-acetylglucosamine (PNAG), poly-N-succinyl glucosamine (PNSG),Wall Teichoic Acid (WTA), Lipoteichoic acid (LTA), a fragment of any ofthe immunogenic carbohydrates, and a combination of two or more of theimmunogenic carbohydrates. In certain aspects, the immunogeniccarbohydrate is conjugated to the oligopeptide.

Further provided for is an isolated polynucleotide comprising a nucleicacid that encodes an attenuated SEA toxoid polypeptide describedelsewhere herein or a multivalent oligopeptide described elsewhereherein. In certain aspects, the polynucleotide comprises the nucleotidesequence SEQ ID NO: 8. The polynucleotide can further comprise aheterologous nucleic acid. In certain aspects, the heterologous nucleicacid comprises a promoter operably associated with the nucleic acidencoding the oligopeptide. Also provided for is a vector comprising thepolynucleotide. In certain aspects, the vector is a plasmid. Alsoprovided for is a host cell comprising the vector. In certain aspects,the host cell is a bacterium, an insect cell, a mammalian cell, or aplant cell. In certain aspects, the bacterium is Escherichia coli.

Further provided is a method of producing a multivalent oligopeptide. Incertain aspects, the method comprises culturing a host cell describedelsewhere herein and recovering the oligopeptide.

Further provided is a composition, such as a therapeutic, immunogenic,and/or antigenic composition, comprising an attenuated SEA toxoid ormultivalent oligopeptide described elsewhere herein, or any combinationthereof, and a carrier. The composition can further comprise anadjuvant. In certain aspects, the adjuvant is alum, aluminum hydroxide,aluminum phosphate, or a glucopyranosyl lipid A-based adjuvant. Thecomposition can also further comprise an additional immunogen. Incertain aspects, the additional immunogen is a bacterial antigen. Incertain aspects, the bacterial antigen is selected from the groupconsisting of a pore forming toxin, a superantigen, a cell surfaceprotein, a fragment of any of the bacterial antigens, and a combinationof two or more of the bacterial antigens.

Further provided is a method of inducing a host immune response againstStaphylococcus aureus. In certain aspects, the method comprisesadministering to a subject in need of the immune response an effectiveamount of an immunogenic or antigenic composition described elsewhereherein. In certain aspects, the immune response is selected from thegroup consisting of an innate response, a humoral response, an antibodyresponse, a cellular response, and a combination of two or more of theimmune responses. In certain aspects, the immune response is an antibodyresponse.

Further provided is a method of preventing or treating a Staphylococcaldisease or infection in a subject. In certain aspects, the methodcomprises administering to a subject in need thereof a compositiondescribed elsewhere herein. In certain aspects, the infection is alocalized or systemic infection of skin, soft tissue, blood, or anorgan, or is auto-immune in nature. In certain aspects, the disease is arespiratory disease, for example, pneumonia. In certain aspects, thedisease is sepsis.

A subject in any of the methods disclosed herein can be a mammal. Incertain aspects, the mammal is a human. In certain aspects, the mammalis bovine or canine.

A composition for administration in any of the methods disclosed hereincan be administered via intramuscular injection, intradermal injection,intraperitoneal injection, subcutaneous injection, intravenousinjection, oral administration, mucosal administration, intranasaladministration, or pulmonary administration.

Further provided for is a composition for use in inducing a host immuneresponse against Staphylococcus aureus in a subject. Further providedfor is a composition for use in preventing or treating a Staphylococcaldisease or infection in a subject. Further provided for is a method ofproducing a vaccine against S. aureus infection. In certain aspects, themethod comprises isolating an attenuated SEA toxoid described elsewhereherein, a multivalent oligopeptide described elsewhere herein, or anycombination thereof, and combining the toxoid, oligopeptide, or anycombination thereof, with an adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 is a schematic of rTBA and rTBA225 constructs. Additionalpotential configurations of the fusion peptide are also shown. Linker:three repeats of the linker GGGGS (4GS) (SEQ ID NO: 15).

FIG. 2A-B illustrates purification of rTBA. FIG. 2A) Process forpurification of rTBA and rTBA 225. FIG. 2B) SDS-PAGE analysis of rTBA.

FIG. 3 shows the comparative immunogenicity of rTBA versus a cocktail ofthe three individual toxoids in mice. A) ELISA and toxin neutralizationassay (TNA) were performed on pooled sera from 5 immunized mice pergroup for the three SAgs. B) Groups of 10 mice were immunized 3 timeswith rTBA formulated either in CpG or Alhydrogel and immunogenicity wasdetermined in ELISA and TNA assays. Data shown are ELISA EC₅₀ and TNANT₅₀ values.

FIG. 4 shows rTBA and rTBA225 safety profiles. Response of human PBMCfrom three donors to SEA, rTBA, SEAH225A, rSEA225, and rTBA225.

FIG. 5 shows the comparative immunogenicity of rTBA225 versus rTBAversus a cocktail of the three individual toxoids in mice. ELISA andtoxin neutralization assay (TNA) were performed on individual sera from10 immunized mice per group for SEA, SEB and TSST-1. Data shown areELISA EC₅₀ (A) and TNA NT₅₀ values (B). TNA to test forcross-neutralization against other super antigens were also performed onpooled sera from the immunized mice. Data shown is percentageneutralization at 1:40 serum dilution (C). Error bars represent standarderrors of mean and the asterisks show statistical difference betweenrTBA225 and SAg cocktail immunized mice sera as determined by theMann-Whitney non-parametric test.

FIG. 6 shows adsorption of rTSST-1 (A) and rTBA225 (B) by Alhydrogel.The proteins were incubated alone (left lanes) or with Alhydrogel at theindicated ratios (protein: Alhydrogel) for 30 minutes at roomtemperature. Following the incubation the samples were centrifuged toprecipitate the adsorbed protein. The supernatant was then subjected toSDS-PAGE analysis and visualized by Coomassie staining. Lack ofdetectable protein band indicates binding to Alhydrogel.

FIG. 7 shows protection provided by rTBA225 against toxin challenge.Groups of 10 mice were vaccinated three times with BSA as a control orrTBA225 formulated in Alhydrogel and challenged with the indicated dosesof wild-type TSST-1, SEA, or SEA. Animals were monitored for 5 days formortality and morbidity.

DETAILED DESCRIPTION I. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a polynucleotide,” is understood torepresent one or more polynucleotides. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term and/or” as used in a phrase such as “Aand/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; Aand B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Systhme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects orembodiments of the disclosure, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

Wherever aspects or embodiments are described with the language“comprising,” otherwise analogous aspects or embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Amino acids are referred to herein by their commonly known three lettersymbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, are referredto by their commonly accepted single-letter codes.

The terms “nucleic acid” or “nucleic acid fragment” refers to any one ormore nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide or construct. Two or more nucleic acids of the disclosurecan be present in a single polynucleotide construct, e.g., on a singleplasmid, or in separate (non-identical) polynucleotide constructs, e.g.,on separate plasmids. Furthermore, any nucleic acid or nucleic acidfragment can encode a single polypeptide, e.g., a single antigen,cytokine, or regulatory polypeptide, or can encode more than onepolypeptide, e.g., a nucleic acid can encode two or more polypeptides.In addition, a nucleic acid can encode a regulatory element such as apromoter or a transcription terminator, or can encode a specializedelement or motif of a polypeptide or protein, such as a secretory signalpeptide or a functional domain.

The term “polynucleotide” is intended to encompass a singular nucleicacid or nucleic acid fragment as well as plural nucleic acids or nucleicacid fragments, and refers to an isolated molecule or construct, e.g., avirus genome (e.g., a non-infectious viral genome), messenger RNA(mRNA), plasmid DNA (pDNA), or derivatives of pDNA (e.g., minicircles asdescribed in (Darquet, A-M et al., Gene Therapy 4:1341-1349, 1997)comprising a polynucleotide. A polynucleotide can be provided in linear(e.g., mRNA), circular (e.g., plasmid), or branched form as well asdouble-stranded or single-stranded forms. A polynucleotide can comprisea conventional phosphodiester bond or a non-conventional bond (e.g., anamide bond, such as found in peptide nucleic acids (PNA)).

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and comprisesany chain or chains of two or more amino acids. Thus, as used herein, a“peptide,” an “oligopeptide,” a “dipeptide,” a “tripeptide,” a“protein,” an “amino acid chain,” an “amino acid sequence,” “a peptidesubunit,” or any other term used to refer to a chain or chains of two ormore amino acids, are included in the definition of a “polypeptide,”(even though each of these terms can have a more specific meaning) andthe term “polypeptide” can be used instead of, or interchangeably withany of these terms. The term further includes polypeptides which haveundergone post-translational modifications, for example, glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, or modification bynon-naturally occurring amino acids.

The term “multivalent oligopeptide” as used herein refers to a fusionprotein comprising two or more attenuated staphylococcal proteins, e.g.,superantigen (SAg) toxoids or any fragments, variants, or derivativesthereof fused together as a single polypeptide in any order. Anoligopeptide can include other heterologous peptides as describedelsewhere herein. Other peptides for inclusion in a multivalentoligopeptide provided herein include various other staphylococcaltoxoids or fragments, variants, or derivatives thereof, describedelsewhere herein or in PCT Publication Nos. WO 2012/109167A1 and WO2013/082558 A1, which are both incorporated by reference herein in theirentireties.

The collection of toxoids and oligopeptides of fusions of toxoidsprovided by the disclosure are collectively referred to herein as a“multivalent oligopeptide and/or SAg toxoid,” or a “multivalentoligopeptide, SAg toxoid, or any combination thereof.” These collectivereferences are meant to include, without limitation, any one toxoid oroligopeptide as provided herein, or two, three, four, or more toxoids oroligopeptides as provided herein.

The terms “fragment,” “derivative,” or “variant” when referring to amultivalent oligopeptide and/or SAg toxoid of the present disclosureinclude any polypeptide which retains at least some of theimmunogenicity or antigenicity of the source protein or proteins.Fragments of multivalent oligopeptides and/or SAgs as described hereininclude proteolytic fragments, deletion fragments or fragments thatexhibit increased solubility during expression, purification, and/oradministration to an animal. Fragments of multivalent oligopeptidesand/or SAgs as described herein further include proteolytic fragments ordeletion fragments which exhibit reduced pathogenicity or toxicity whendelivered to a subject. Polypeptide fragments further include anyportion of the polypeptide which comprises an antigenic or immunogenicepitope of the source polypeptide, including linear as well asthree-dimensional epitopes.

An “epitopic fragment” of a polypeptide is a portion of the polypeptidethat contains an epitope. An “epitopic fragment” can, but need not,contain amino acid sequence in addition to one or more epitopes.

The term “variant,” as used herein, refers to a polypeptide that differsfrom the recited polypeptide due to amino acid substitutions, deletions,insertions, and/or modifications. Non-naturally occurring variants canbe produced using art-known mutagenesis techniques. In some aspects,variant polypeptides differ from an identified sequence by substitution,deletion or addition of three amino acids or fewer. Such variants cangenerally be identified by modifying a polypeptide sequence, andevaluating the antigenic or pathogenic properties of the modifiedpolypeptide using, for example, the representative procedures describedherein. In some aspects, variants of a multivalent oligopeptide and/orSAg toxoid form a protein complex which is less toxic than the wild-typecomplex.

Polypeptide variants disclosed herein exhibit at least about 85%, 90%,94%, 95%, 96%, 97%, 98%, 99% or 99.9% sequence identity with identifiedpolypeptide. Variant polypeptides can comprise conservative ornon-conservative amino acid substitutions, deletions or insertions.Variants can comprise multivalent oligopeptides and/or SAgs identical tothe various wild-type staphylococcal proteins except for at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more amino acid substitutions,including specific mutations described elsewhere herein, where thesubstitutions render complex less toxic than a corresponding wild-typeprotein complex. Derivatives of multivalent oligopeptides and/or SAgs asdescribed herein are polypeptides which have been altered so as toexhibit additional features not found on the native polypeptide.Examples include fusion proteins. An analog is another form of amultivalent oligopeptide and/or SAg toxoid described herein. An exampleis a proprotein which can be activated by cleavage of the proprotein toproduce an active mature polypeptide.

Variants can also, or alternatively, contain other modifications,whereby, for example, a polypeptide can be conjugated or coupled, e.g.,fused to a heterologous amino acid sequence, e.g., a signal (or leader)sequence at the N-terminal end of the protein which co-translationallyor post-translationally directs transfer of the protein. The polypeptidecan also be conjugated or produced coupled to a linker or other sequencefor ease of synthesis, purification or identification of the polypeptide(e.g., 6-His), or to enhance binding of the polypeptide to a solidsupport. For example, the polypeptide can be conjugated or coupled to animmunoglobulin Fc region. The polypeptide can also be conjugated orcoupled to a sequence that imparts or modulates the immune response tothe polypeptide (e.g., a T-cell epitope, B-cell epitope, cytokine,chemokine, etc.) and/or enhances uptake and/or processing of thepolypeptide by antigen presenting cells or other immune system cells.The polypeptide can also be conjugated or coupled to otherpolypeptides/epitopes from Staphylococcus sp. and/or from other bacteriaand/or other viruses to generate a hybrid immunogenic protein that aloneor in combination with various adjuvants can elicit protective immunityto other pathogenic organisms. The polypeptide can also be conjugated orcoupled to moieties which confer greater stability or improve half lifesuch as, but not limited to albumin, an immunoglobulin Fc region,polyethylene glycol (PEG), and the like. The polypeptide can also beconjugated or coupled to moieties (e.g., immunogenic carbohydrates,e.g., a capsular polysaccharide or a surface polysaccharide) fromStaphylococcus sp. and/or from other bacteria and/or other viruses togenerate a modified immunogenic protein that alone or in combinationwith one or more adjuvants can enhance and/or synergize protectiveimmunity. In certain aspects, the polypeptide described herein furthercomprises an immunogenic carbohydrate. In one aspect, the immunogeniccarbohydrate is a saccharide.

The term “saccharide” throughout this specification can indicatepolysaccharide or oligosaccharide and includes both. Polysaccharides ofthe disclosure can be isolated from bacteria and can be sized by knownmethods. For example, full length polysaccharides can be “sized” (e.g.,their size can be reduced by various methods such as acid hydrolysistreatment, hydrogen peroxide treatment, sizing by EMULSIFLEX® followedby a hydrogen peroxide treatment to generate oligosaccharide fragmentsor microfluidization). Polysaccharides can be sized in order to reduceviscosity in polysaccharide samples and/or to improve filterability forconjugated products. Oligosaccharides have a low number of repeat units(e.g., 5-30 repeat units) and are typically hydrolyzed polysaccharides.Polysaccharides of the disclosure can be produced recombinantly.

S. aureus capsular antigens are surface associated, limited in antigenicspecificity, and highly conserved among clinical isolates. In oneaspect, the immunogenic carbohydrate of the disclosure is a capsularpolysaccharide (CP) of S. aureus. In one aspect, a capsular saccharidecan be a full length polysaccharide, however in other aspects it can beone oligosaccharide unit, or a shorter than native length saccharidechain of repeating oligosaccharide units. Serotyping studies ofstaphylococcal isolates have revealed several putative capsularserotypes, with types 5 and 8 (CP5 and CP8) being the most prevalentamong isolates from clinical infections, accounting for about 25% and50% of isolates recovered from humans, respectively (O'Riordan and Lee,Clinical Microbiology Reviews, January 2004, p. 218-234, Vol. 17, No. 1;Poutrel and Sutra, J Clin Microbiol. 1993 February; 31(2):467-9). Thesame isolates were also recovered from poultry, cows, horses and pigs(Tollersrud et al., J Clin Microbiol. 2000 August; 38(8):2998-3003;Cunnion K M et al., Infect Immun. 2001 November; 69(11):6796-803). Type5 and 8 capsular polysaccharides purified from the prototype strainsReynolds and Becker, respectively, are structurally very similar to eachother and to the capsule made by strain T, described previously by Wuand Park (Wu and Park. 1971. J. Bacteriol. 108:874-884). Type 5 has thestructure (→4)-3-O—Ac—ß-D-ManNAcA-(1→4)-α-L-FucNAc-(1→3)-ß-D-FucNAc-(1→)n (Fournier, J. M., et al.,1987. Ann. Inst. Pasteur Microbiol. 138:561-567; Moreau, M., et al.,1990. Carbohydr. Res. 201.285-297), and type 8 has the structure(→3)-4-O—Ac—ß-D-ManNAcA-(1→3)α-L-FucNAc-(1→3)-ß-D-FucNAc-(1→)_(n)(Fournier, J. M., et al., 1984. Infect. Immun. 45:87-93). Type 5 and 8polysaccharides differ only in the linkages between the sugars and inthe sites of O-acetylation of the mannosaminuronic acid residues, yetthey are serologically distinct.

Type 5 and 8 CP conjugated to a detoxified recombinant Pseudomonasaeruginosa exotoxin A carrier were shown to be highly immunogenic andprotective in a mouse model (A Fattom et al., Infect Immun. 1993 March;61(3): 1023-1032; A Fattom et al., Infect Immun. 1996 May; 64(5):1659-1665) and passive transfer of the CP5-specific antibodies from theimmunized animals induced protection against systemic infection in mice(Lee et al., Infect Immun. 1997 October; 65(10): 4146-4151) and againstendocarditis in rats challenged with a serotype 5 S. aureus (ShinefieldH et al., N Engl J Med. 2002 Feb. 14; 346(7):491-6). A bivalent CP5 andCP8 conjugate vaccine (StaphVAX®, Nabi Biopharmaceutical) was developedthat provided 75% protection in mice against S. aureus challenge. Thevaccine has been tested on humans (Fattom A I et al., Vaccine. 2004 Feb.17; 22(7):880-7; Maira-Litrán T et al., Infect Immun. 2005 October;73(10):6752-62). In certain aspects, the recombinant peptide ormultivalent oligopeptide of the disclosure is combined with orconjugated to an immunogenic carbohydrate (e.g., CP5, CP8, a CP fragmentor a combination thereof).

Immunization with poly-N-acetylglucosamine (PNAG) (McKenney D. et al.,Science. 1999 May 28; 284(5419):1523-7) or poly-N-succinyl glucosamine(PNSG) (Tuchscherr L P. et al., Infect Immun. 2008 December;76(12):5738-44. Epub 2008 Sep. 22), both S. aureus surfacecarbohydrates, has been shown to generate at least partial protectionagainst S. aureus challenge in experimental animal models. PNSG wasidentified as the chemical form of the S. epidermidis capsularpolysaccharide/adhesin (PS/A) which mediates adherence ofcoagulase-negative staphylococci (CoNS) to biomaterials, serves as thecapsule for strains of CoNS that express PS/A, and is a target forprotective antibodies. PNSG is also made by S. aureus, where it is anenvironmentally regulated, in vivo-expressed surface polysaccharide andsimilarly serves as a target for protective immunity (McKenney D. etal., J. Biotechnol. 2000 Sep. 29; 83(1-2): 37-44). In certain aspects ofthe disclosure, the immunogenic carbohydrate is a surfacepolysaccharide, e.g., poly-N-acetylglucosamine (PNAG), poly-N-succinylglucosamine (PNSG), a surface polysaccharide fragment or a combinationthereof.

Wall Teichoic Acid (WTA) is a prominent polysaccharide widely expressedon S. aureus strains (Neuhaus, F. C. and J. Baddiley, Microbiol Mol BiolRev, 2003. 67(4):686-723) and antisera to WTA have been shown to induceopsonophagocytic killing alone and in presence of complement ((Thakker,M., et al., Infect Immun, 1998. 66(11):5183-9), and Falom et al, U.S.Pat. No. 7,754,225). WTA is linked to peptidoglycans and protrudesthrough the cell wall becoming prominently exposed on non-encapsulatedstrains such as USA300 responsible for most cases of community acquiredMRSA (CA MRSA) in the US (Hidron, A. I., et al., Lancet Infect Dis,2009. 9(6):384-92).

Lipoteichoic acid (LTA) is a constituent of the cell wall ofGram-positive bacteria, e.g., Staphylococcus aureus. LTA can bind totarget cells non-specifically through membrane phospholipids, orspecifically to CD14 and to Toll-like receptors. Target-bound LTA caninteract with circulating antibodies and activate the complement cascadeto induce a passive immune kill phenomenon. It also triggers the releasefrom neutrophils and macrophages of reactive oxygen and nitrogenspecies, acid hydrolases, highly cationic proteinases, bactericidalcationic peptides, growth factors, and cytotoxic cytokines, which canact in synergy to amplify cell damage.

In certain aspects, a surface polysaccharide is combined with orconjugated to a polypeptide of the disclosure. In certain aspects thesurface polysaccharide is, e.g., poly-N-acetylglucosamine (PNAG),poly-N-succinyl glucosamine (PNSG), Wall Teichoic Acid (WTA),Lipoteichoic acid (LPA), a fragment of any of said surfacepolysaccharides, or a combination of two or more of said surfacepolysaccharides.

The term “sequence identity” as used herein refers to a relationshipbetween two or more polynucleotide sequences or between two or morepolypeptide sequences. When a position in one sequence is occupied bythe same nucleic acid base or amino acid in the corresponding positionof the comparator sequence, the sequences are said to be “identical” atthat position. The percentage “sequence identity” is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid occurs in both sequences to yield the number of“identical” positions. The number of “identical” positions is thendivided by the total number of positions in the comparison window andmultiplied by 100 to yield the percentage of “sequence identity.”Percentage of “sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window and a homologouspolypeptide from another isolate. In order to optimally align sequencesfor comparison, the portion of a polynucleotide or polypeptide sequencein the comparison window can comprise additions or deletions termed gapswhile the reference sequence is kept constant. An optimal alignment isthat alignment which, even with gaps, produces the greatest possiblenumber of “identical” positions between the reference and comparatorsequences. Percentage “sequence identity” between two sequences can bedetermined using the version of the program “BLAST 2 Sequences” which isavailable from the National Center for Biotechnology Information as ofSep. 1, 2004, which program incorporates the programs BLASTN (fornucleotide sequence comparison) and BLASTP (for polypeptide sequencecomparison), which programs are based on the algorithm of Karlin andAltschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). Whenutilizing “BLAST 2 Sequences,” parameters that were default parametersas of Sep. 1, 2004, can be used for word size (3), open gap penalty(11), extension gap penalty (1), gap drop-off (50), expect value (10)and any other required parameter including but not limited to matrixoption.

The term “epitope,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, for example amammal, for example, a human. An “immunogenic epitope,” as used herein,is defined as a portion of a protein that elicits an immune response inan animal, as determined by any method known in the art. The term“antigenic epitope,” as used herein, is defined as a portion of aprotein to which an antibody or T-cell receptor can immunospecificallybind its antigen as determined by any method well known in the art.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Whereas allimmunogenic epitopes are antigenic, antigenic epitopes need not beimmunogenic.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it can beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, and the like, are outside the coding region.

The term “codon optimization” is defined herein as modifying a nucleicacid sequence for enhanced expression in the cells of the host ofinterest by replacing at least one, more than one, or a significantnumber, of codons of the native sequence with codons that are morefrequently or most frequently used in the genes of that host. Variousspecies exhibit particular bias for certain codons of a particular aminoacid.

The terms “composition” or “pharmaceutical composition” can includecompositions containing immunogenic polypeptides of the disclosure alongwith e.g., adjuvants or pharmaceutically acceptable carriers,excipients, or diluents, which are administered to an individual alreadysuffering from S. aureus infection or an individual in need ofimmunization against S. aureus infection.

The term “pharmaceutically acceptable” refers to compositions that are,within the scope of sound medical judgment, suitable for contact withthe tissues of human beings and animals without excessive toxicity orother complications commensurate with a reasonable benefit/risk ratio.In some aspects, the polypeptides, polynucleotides, compositions, andvaccines described herein are pharmaceutically acceptable.

An “effective amount” is that amount the administration of which to anindividual, either in a single dose or as part of a series, is effectivefor treatment or prevention. An amount is effective, for example, whenits administration results in a reduced incidence of S. aureus infectionrelative to an untreated individual, as determined, e.g., afterinfection or challenge with infectious S. aureus, including, but is notlimited to reduced bacteremia, reduced toxemia, reduced sepsis, reducedsymptoms, increased immune response, modulated immune response, orreduced time required for recovery. This amount varies depending uponthe health and physical condition of the individual to be treated, thetaxonomic group of individual to be treated (e.g., human, nonhumanprimate, primate, etc.), the responsive capacity of the individual'simmune system, the extent of treatment or protection desired, theformulation of the vaccine, a professional assessment of the medicalsituation, and other relevant factors. It is expected that the effectiveamount will fall in a relatively broad range that can be determinedthrough routine trials. Typically a single dose is from about 10 μg to10 mg/kg body weight of purified polypeptide or an amount of a modifiedcarrier organism or virus, or a fragment or remnant thereof, sufficientto provide a comparable quantity of recombinantly expressed multivalentoligopeptide and/or SAg toxoid as described herein. The term “peptidevaccine” or “subunit vaccine” refers to a composition comprising one ormore polypeptides described herein, which when administered to an animalare useful in stimulating an immune response against staphylococcal(e.g., S. aureus) infection.

The term “subject” is meant any subject, particularly a mammaliansubject, for whom diagnosis, prognosis, immunization, or therapy isdesired. Mammalian subjects include, but are not limited to, humans,domestic animals, farm animals, zoo animals such as bears, sportanimals, pet animals such as dogs, cats, guinea pigs, rabbits, rats,mice, horses, cattle, bears, cows; primates such as apes, monkeys,orangutans, and chimpanzees; canids such as dogs and wolves; felids suchas cats, lions, and tigers; equids such as horses, donkeys, and zebras;food animals such as cows, pigs, and sheep; ungulates such as deer andgiraffes; rodents such as mice, rats, hamsters and guinea pigs; and soon. In one aspect, the subject is a human subject.

As used herein, a “subject in need thereof” refers to an individual forwhom it is desirable to treat, i.e., to prevent, cure, retard, or reducethe severity of staphylococcal (e.g., S. aureus) disease symptoms, orresult in no worsening of disease cause by S. aureus over a specifiedperiod of time, or both.

The terms “priming” or “primary” and “boost” or “boosting” as usedherein refer to the initial and subsequent immunizations, respectively,i.e., in accordance with the definitions these terms normally have inimmunology. However, in certain aspects, e.g., where the primingcomponent and boosting component are in a single formulation, initialand subsequent immunizations are not be necessary as both the “prime”and the “boost” compositions are administered simultaneously.

As used herein, “superantigenic activity” is a measure of a multivalentoligopeptide's or SAg toxoid's residual toxicity and can be measured incomparison to that of a wild-type SAg toxin or to another reference SAgtoxoid or SAg toxoid containing multivalent oligopeptide. For purposesof this disclosure, an increase or decrease in “superantigenic activity”in comparison to a reference polypeptide can be determined by measuringthe activity of a SAg toxin, toxoid, or oligopeptide against isolatedperipheral blood mononuclear cells (PBMCs) in an in vitro stimulationassay as described elsewhere herein.

II. Superantigen (SAg) Toxoids and Multivalent Oligopeptides

This disclosure provides for recombinant oligopeptide fusion proteinscomprised of attenuated polypeptide subunits, referred to herein as“toxoids,” derived from Staphylococcal superantigens. In certainaspects, the SAg toxoid is attenuated by one or more mutations todecrease its superantigenic activity, toxicity, and/or virulence, whilemaintaining its immunogenicity. Accordingly, this disclosure providesfor an attenuated Staphylococcus aureus-derived superantigen (SAg)Staphylococcal enterotoxin A (SEA) toxoid or fragment, variant, orderivative thereof, comprising four mutations relative to wild-type SEAcorresponding to L48R, D70R, Y92A, and H225A mutations in SEQ ID NO: 4.In certain aspects, the attenuated SEA toxoid or fragment, variant, orderivative thereof, having the four specified mutations, comprises anamino acid sequence that is at least 90% identical to SEQ ID NO: 4. Incertain aspects, the attenuated SEA toxoid or fragment, variant, orderivative thereof comprises and/or consists of SEQ ID NO: 4. It will beunderstood that the nomenclature used herein to describe point mutations(e.g. “L48R”) are in comparison to wild-type SAg proteins which do notcontain the N-terminal Methionine that was required for heterologousexpression.

In certain aspects, the SEA toxoid or fragment, variant, or derivativesthereof, having the four specified mutations, has decreasedsuperantigenic activity, decreased toxicity, and/or is less virulentthan a wild-type SEA toxin. In certain aspects, the SEA toxoid orfragment, variant, or derivatives thereof, having the four specifiedmutations, has decreased superantigenic activity, decreased toxicity,and/or is less virulent than a SEA toxoid comprising SEQ ID NO: 3(SEA_(L48R/D70R/Y92A)). In certain aspects, the SEA toxoid or fragment,variant, or derivatives thereof, having the four specified mutations,has decreased superantigenic activity, decreased toxicity, and/or isless virulent than a SEA toxoid consisting of SEQ ID NO: 3.

In certain aspects, the attenuated SEA toxoid or fragment, variant, orderivative thereof, having the four specified mutations, has less than50%, less than 40%, less than 30%, less than 20%, less than 10%, lessthan 5%, less than 3%, less than 2%, or less than 1% of thesuperantigenic activity of a wild-type SEA toxin. In certain aspects,the attenuated SEA toxoid or fragment, variant, or derivative thereof,having the four specified mutations, has less than 50%, less than 40%,less than 30%, less than 20%, less than 10%, less than 5%, less than 3%,less than 2%, or less than 1% of the superantigenic activity of a SEAtoxoid comprising SEQ ID NO: 3. In certain aspects, the attenuated SEAtoxoid or fragment, variant, or derivative thereof, having the fourspecified mutations, has less than 50%, less than 40%, less than 30%,less than 20%, less than 10%, less than 5%, less than 3%, less than 2%,or less than 1% of the superantigenic activity of a SEA toxoidconsisting of SEQ ID NO: 3.

In certain aspects of any of the attenuated SEA toxoids or fragments,variants, or derivatives thereof, comprising four mutations relative towild-type SEA corresponding to the L48R, D70R, Y92A, and H225A mutationsin SEQ ID NO: 4 as disclosed herein, immunogenicity is maintained ascompared to a wild-type SEA toxin, a SEA toxoid comprising SEQ ID NO: 3,and/or a SEA toxoid consisting of SEQ ID NO: 3. In certain aspects,immunization with the SEA toxoid or fragment, variant, or derivativethereof, comprising the four specified mutations, elicits neutralizingantibodies against a wild-type SEA toxin.

Further, in certain aspects, this disclosure provides a multivalentoligopeptide comprising a fusion of two or more, e.g., two, three, four,five, six, seven, eight, nine, ten or more Staphylococcus aureus-derivedtoxoids or fragments, variants, or derivatives thereof arranged in anyorder. The two or more Staphylococcus aureus-derived toxoids orfragments, variants, or derivatives thereof of the multivalentoligopeptide can be the same or different.

U.S. Publication No. 2016/0185829 A1 (incorporated herein by reference)describes a simplified Superantigen (SAg) toxoid vaccine comprising afusion oligopeptide of mutants of Superantigens, namely recombinantTSST-1_(L30R/D27A/I46A) (SEQ ID NO: 1), SEB_(L45R/Y89A/Y94A) (SEQ ID NO:2), and SEA_(L48R/D70R/Y92A) (SEQ ID NO: 3). This multivalentoligopeptide is referred to herein as rTBA (FIG. 1) and has the aminoacid sequence SEQ ID NO: 5. The rTBA construct was capable of inducingbroad neutralizing antibodies. This fusion protein induced a bettertotal antibody and neutralizing response compared to a simple mixture ofthe three individual toxoids, but it retained some residualsuperantigenic activity.

Provided herein is a multivalent oligopeptide that improves upon rTBA.In certain aspects, the multivalent oligopeptide comprises a fusionprotein of two or more SAg toxoids having reduced superantigenicactivity, toxicity, and/or virulence relative to a SAg fusion proteincomprising and/or consisting of SEQ ID NO: 5. In certain aspects, themultivalent oligopeptide has less than 50%, less than 40%, less than30%, less than 20%, less than 10%, less than 5%, less than 3%, less than2%, or less than 1% of the superantigenic activity, toxicity, and/orvirulence of a wild-type SEA toxin and/or a SAg fusion proteincomprising SEQ ID NO: 5 (FIG. 4). In certain aspects, the multivalentoligopeptide maintains the immunogenicity of the SAg fusion proteincomprising and/or consisting of SEQ ID NO: 5. In certain aspects,immunization with the multivalent oligopeptide elicits neutralizingantibodies against a SAg TSST-1 toxin, a SAg SEB toxin, a SAg SEA toxin,or any combination thereof. In certain aspects, immunization with themultivalent oligopeptide elicits neutralizing antibodies to SAg toxinsother than TSST-1, SEB, or SEA. In certain aspects, the multivalentoligopeptide exhibits greater and/or broader immunogenicity than anequimolar cocktail of the individual SAg toxoids from which it iscomposed (FIG. 5). In certain aspects, immunization of a subject withthe multivalent oligopeptide provides protection against at least one ormore of wild-type SAg TSST-1 toxin, wild-type SAg SEB toxin, andwild-type SAg SEA toxin (FIG. 7). In certain aspects, the multivalentoligopeptide or a composition comprising the oligopeptide can be used totreat or prevent a Staphylococcal disease or infection.

In certain aspects of this disclosure, a multivalent oligopeptideincludes a staphylococcal SAg toxoid or fragment, variant, or derivativethereof including, without limitation, a toxoid derivative ofstaphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB),staphylococcal enterotoxins C1-3 (SEC1-3), staphylococcal enterotoxin E(SEE), staphylococcal enterotoxin H (SHE), staphylococcal enterotoxin I(SEI), staphylococcal enterotoxin K (SEK), staphylococcal toxic shocksyndrome toxin-1 (TSST-1), streptococcal pyrogenic exotoxin C (SpeC),staphylococcal enterotoxin D (SED), streptococcal pyrogenic exotoxin A(SpeA), or any combination thereof, in any order.

In certain aspects, the multivalent oligopeptide includes astaphylococcal toxic shock syndrome toxin-1 (TSST-1) toxoid or fragment,variant, or derivative thereof. In certain aspects, the TSST-1 toxoid isthe attenuated toxoid TSST-1_(L30R/D27A/I46A) (SEQ ID NO: 1), or aTSST-1 toxoid comprising the three attenuating mutations relative towild-type TSST-1 corresponding to the L30R, D27A, and I46A mutations inSEQ ID NO: 1 and an amino acid sequence at least 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In certainaspects, the oligopeptide includes a staphylococcal enterotoxin B (SEB)toxoid or fragment, variant, or derivative thereof. In certain aspects,the SEB toxoid is the attenuated toxoid SEB_(L45R/Y89A/Y94A) (SEQ ID NO:2), or a SEB toxoid comprising the three attenuating mutations relativeto wild-type SEB corresponding to the L45R, Y89A, and Y94A mutations inSEQ ID NO: 2 and an amino acid sequence at least 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In certainaspects, the oligopeptide includes a staphylococcal enterotoxin A (SEA)toxoid or fragment, variant, or derivative thereof. In certain aspects,the SEA toxoid is the attenuated toxoid SEAL48RjD70RY92AH225A (SEQ IDNO: 4), or an SEA toxoid comprising the four attenuating mutationsrelative the wild-type SEA corresponding to the L48R, D70R, Y92A, andH225A mutations in SEQ ID NO: 4 and an amino acid sequence at least 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4.

In certain aspects, a multivalent oligopeptide as provided hereincomprises at least one Staphylococcal enterotoxin A (SEA) attenuatedtoxoid comprising four mutations relative to wild-type SEA correspondingto the L48R, D70R, Y92A, and H225A mutations inSEA_(L48R/D70R/Y92A/H225A) (SEQ ID NO: 4) as described elsewhere herein.In certain aspects, the multivalent oligopeptide comprises two or moreor three or more SAg toxoids or fragments, variants, or derivativesthereof. In certain aspects, the oligopeptide further comprises astaphylococcal enterotoxin B (SEB) attenuated toxoid as describedelsewhere herein, a staphylococcal toxic shock syndrome toxin-1 (TSST-1)attenuated toxoid as described elsewhere herein, and any combinationthereof. In certain aspects, the TSST-1 toxoid comprises three mutationsrelative to wild-type TSST-1 corresponding to the L30R, D27A, and I46Amutations in SEQ ID NO: 1 and an amino acid sequence at least 90%identical to SEQ ID NO: 1; the SEB toxoid comprises three mutationsrelative to wild-type SEB corresponding to the L45R, Y89A, and Y94Amutations in SEQ ID NO: 2 and an amino acid sequence at least 90%identical to SEQ ID NO: 2; and the SEA attenuated toxoid comprises fourmutations relative to wild-type SEA corresponding to the L48R, D70R,Y92A, and H225A mutations in the SEA toxoid of SEQ ID NO: 4 and an aminoacid sequence at least 90% identical to SEQ ID NO: 4. In certainaspects, the TSST-1 toxoid comprises the amino acid sequence SEQ ID NO:1; the SEB toxoid comprises the amino acid sequence of SEQ ID NO: 2; andthe SEA attenuated toxoid comprises the amino acid sequence SEQ ID NO:4.

In certain aspects, the multivalent oligopeptide includes the SAgattenuated toxoids SEB_(L45R/Y89A/Y94A) (“B”),SEA_(L48R/D70R/Y92A/H225A) (“A225”), TSST-1_(L30R/D27A/I46A) (“T”), orany combination thereof. In certain aspects, the multivalentoligopeptide includes at least SEA_(L48R/D70R/Y92A/H225A). In certainaspects, the multivalent oligopeptide comprises, consists of, orconsists essentially of a “TBA225” fusion (rTBA225; SEQ ID NO: 6), whichis a fusion of TSST-1_(L30R/D27A/I46A), SEB_(L45R/Y89A/Y94A), andSEA_(L48R/D70R/Y92A/H225A), in that order. Or the oligopeptide has theattenuating mutations corresponding to those of SEB_(L45R/Y89A/Y94A),SEA_(L48R/D70R/Y92A/H225A), and TSST-1_(L30R/D27A/I46A), wherein theoligopeptide comprises, consists, or consists essentially of an aminoacid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 6.

As noted, in certain aspects, the multivalent oligopeptide is rTBA225(SEQ IN NO: 6), which is a fusion of the SAg triple mutantsTSST-1_(L30R/D27A/Y46A) (SEQ ID NO: 1) and SEB_(L45R/Y89A/Y94A) (SEQ IDNO: 2) and SEA quadruple mutant SEA_(L48R/D70R/Y92A/H225A) (SEQ ID NO:4). rTBA225 retains the superior immunogenicity of rTBA (SEQ ID NO: 5)while having reduced superantigenic activity. Additional possibleconfigurations with different orderings of the aforementioned SAgtoxoids are shown in FIG. 1. Also provided for in this disclosure is amethod for tag-free purification of rTBA and rTBA225.

The SAg toxoids can be linked together in any order, either with ourwithout linkers, and can be the same or different. In some aspects, theSAg toxoids included in the multivalent oligopeptide can be directlyfused to each other. In other aspects, the SAg toxoids included in themultivalent oligopeptide can be associated via a linker. Suitablelinkers can be chosen based on their ability to adopt a flexible,extended conformation, or a secondary structure that can interact withjoined epitopes, or based on their ability to increase overallsolubility of the fusion polypeptide, or based on their lack ofelectrostatic or water-interaction effects that influence joined peptideregions. In certain aspects, the linker is a peptide linker. In certainaspects, a peptide linker for use in a multivalent oligopeptide asprovided herein can include at least one, but no more than 50 aminoacids, e.g., small amino acids that provide a flexible chain, e.g.,glycine, serine, alanine, or a combination thereof. In certain aspects,a linker for use in a multivalent oligopeptide as provided herein caninclude (GGGS)_(n) (SEQ ID NO: 13) or (GGGGS)_(n)(SEQ ID NO: 14),wherein n is a integer from 1 to 10. In certain aspects, such as in thefusion peptide rTBA225 (SEQ ID NO: 6), the linker is a (GGGGS)_(n)linker in which n=3 (SEQ ID NO: 15).

In certain aspects the multivalent oligopeptide comprises, consists of,or consists essentially of the amino acid sequence SEQ ID NO: 6.

TABLE 1 SAgs and Multivalent Oligopeptide Protein Sequences SEQ ID NOTSST-1_(L30R/D27A/I46A) MSTNDNIKDLLDWYSSGSDTFTNSEVL A NS R GSMRIK  1(Mutations relative to NTDGSISLI A FPSPYYSPAFTKGEKVDLNTKRTKKSQHwild-type TSEGTYIHFQISGVTNTEKLPTPIELPLKVKVHGKDSPL bold/underlined)KYWPKFDKKQLAISTLDFEIRHQLTQIHGLYRSSDKTGGYWKITMNDGSTYQSDLSKKFEYNTEKPPINIDEIKT IEAEIN Wild-type TSST-1MSTNDNIKDLLDWYSSGSDTFTNSEVLDNSLGSMRIK  9NTDGSISLIIFPSPYYSPAFTKGEKVDLNTKRTKKSQHTSEGTYIHFQISGVTNTEKLPTPIELPLKVKVHGKDSPLKYWPKFDKKQLAISTLDFEIRHQLTQIHGLYRSSDKTGGYWKITMNDGSTYQSDLSKKFEYNTEKPPINIDEIKTI EAEIN SEB_(L45R/Y89A/Y94A)MESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSA  2 (Mutations INVKSIDQF RYFDLIYSIKDTKLGNYDNVRVEFKNKDL bold/underlined) ADKYKDKYVDVFGAN A YYQC AFSKKTNDINSHQTD KRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVKNKKLYEFNNSPYETGYIKFIENENSFWYDMMPAPGDKFDQSKYLMM YNDNKMVDSKDVKIEVYLTTKKKWild-type SEB MESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSA 10INVKSIDQFLYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKYKDKYVDVFGANYYYQCYFSKKTNDINSHQTD KRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVKNKKLYEFNNSPYETGYIKFIENENSFWYDMMPAPGDKFDQSKYLMM YNDNKMVDSKDVKIEVYLTTKKK SEA_(H225A)EKSEEINEKDLRKKSELQGTALGNLKQIYYYNEKAKT 11 (MutationENKESHDQFLQHTILFKGFFTDHSWYNDLLVDFDSKD bold/underlined)IVDKYKGKKVDLYGAYYGYQCAGGTPNKTACMYGG VTLHDNNRLTEEKKVPINLWLDGKQNTVPLETVKTNKKNVTVQELDLQARRYLQEKYNLYNSDVFDGKVQRGLIVFHTSTEPSVNYDLFGAQGQYSNTLLRIYRDNKTI NSENM A IDIYLYTSSEA_(L48R/D70R/Y92A) EKSEEINEKDLRKKSELQGTALGNLKQIYYYNEKAKT  3 (MutationsENKESHDQF R QHTILFKGFFTDHSWYNDLLV R FDSKD bold/underlined)IVDKYKGKKVDLYGAY A GYQCAGGTPNKTACMYGGVTLHDNNRLTEEKKVPINLWLDGKQNTVPLETVKTN KKNVTVQELDLQARRYLQEKYNLYNSDVFDGKVQRGLIVFHTSTEPSVNYDLFGAQGQYSNTLLRIYRDNKTI NSENMHIDIYLYTSSEA_(L48R/D70R/Y92A/H225A) EKSEEINEKDLRKKSELQGTALGNLKQIYYYNEKAKT  4(Mutations ENKESHDQF R QHTILFKGFFTDHSWYNDLLV R FDSKD bold/underlined)IVDKYKGKKVDLYGAY A GYQCAGGTPNKTACMYGGVTLHDNNRLTEEKKVPINLWLDGKQNTVPLETVKTN KKNVTVQELDLQARRYLQEKYNLYNSDVFDGKVQRGLIVFHTSTEPSVNYDLFGAQGQYSNTLLRIYRDNKTI NSENM A IDIYLYTS Wild-type SEAEKSEEINEKDLRKKSELQGTALGNLKQIYYYNEKAKT 12ENKESHDQFLQHTILFKGFFTDHSWYNDLLVDFDSKDIVDKYKGKKVDLYGAYYGYQCAGGTPNKTACMYGG VTLHDNNRLTEEKKVPINLWLDGKQNTVPLETVKTNKKNVTVQELDLQARRYLQEKYNLYNSDVFDGKVQRGLIVFHTSTEPSVNYDLFGAQGQYSNTLLRIYRDNKTI NSENMHIDIYLYTSrTBA Fusion Protein: MSTNDNIKDLLDWYSSGSDTFTNSEVLANSRGSMRIK  5TSST-1_(L30R/D27A/I46A)- NTDGSISLIAFPSPYYSPAFTKGEKVDLNTKRTKKSQHL-SEB_(L45R/Y89A/Y94A)-L- TSEGTYIHFQISGVTNTEKLPTPIELPLKVKVHGKDSPLSEA_(L48R/D70R/Y92A) KYWPKFDKKQLAISTLDFEIRHQLTQIHGLYRSSDKT(Linkers underlined) GGYWKITMNDGSTYQSDLSKKFEYNTEKPPINIDEIKTIEAEINGGGGSGGGGSGGGGSESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSAINVKSIDQFRYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKYKDKYVDVFGANA YYQCAFSKKTNDINSHQTDKRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVKNKKLYEFNNSPYETGYIKFIENENSEW YDMMPAPGDKFDQSKYLMMYNDNKMVDSKDVKIEVYLTTKKKGGGGSGGGGSGGGGSEKSEEINEKDLRKKSELQGTALGNLKQIYYYNEKAKTENKESHDQFRQHTILFKGFFTDHSWYNDLLVRFDSKDIVDKYKGKKVDLYGAYAGYQCAGGTPNKTACMYGGVTLHDNNRLTEE KKVPINLWLDGKQNTVPLETVKTNKKNVTVQELDLQARRYLQEKYNLYNSDVFDGKVQRGLIVFHTSTEPSVNYDLFGAQGQYSNTLLRIYRDNKTINSENMHIDIYLYTS rTBA225 FusionMSTNDNIKDLLDWYSSGSDTFTNSEVLANSRGSMRIK  6 Protein:NTDGSISLIAFPSPYYSPAFTKGEKVDLNTKRTKKSQH TSST-1_(L30R/D27A/I46A)-TSEGTYIHFQISGVTNTEKLPTPIELPLKVKVHGKDSPL L-SEB_(L45R/Y89A/Y94A)-L-KYWPKFDKKQLAISTLDFEIRHQLTQIHGLYRSSDKT SEA_(L48R/D70R/Y92A/H225A)GGYWKITMNDGSTYQSDLSKKFEYNTEKPPINIDEIKT (Linkers underlined)IEAEINGGGGSGGGGSGGGGSESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSAINVKSIDQFRYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKYKDKYVDVFGANA YYQCAFSKKTNDINSHQTDKRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVKNKKLYEFNNSPYETGYIKFIENENSEW YDMMPAPGDKFDQSKYLMMYNDNKMVDSKDVKIEVYLTTKKKGGGGSGGGGSGGGGSEKSEEINEKDLRKKSELQGTALGNLKQIYYYNEKAKTENKESHDQFRQHTILFKGFFTDHSWYNDLLVRFDSKDIVDKYKGKKVDLYGAYAGYQCAGGTPNKTACMYGGVTLHDNNRLTEE KKVPINLWLDGKQNTVPLETVKTNKKNVTVQELDLQARRYLQEKYNLYNSDVFDGKVQRGLIVFHTSTEPSVNYDLFGAQGQYSNTLLRIYRDNKTINSENMAIDIYLYTS

In another aspect, the multivalent oligopeptide and/or SAg toxoid asprovided herein can be attached to a heterologous polypeptide. Variousheterologous polypeptides can be used, including, but not limited to anN- or C-terminal peptide imparting stabilization, secretion, orsimplified purification, such as a hexa-Histidine-tag, a ubiquitin tag,a NusA tag, a chitin binding domain, ompT, ompA, peiB, DsbA, DsbC,c-myc, KSI, polyaspartic acid, (Ala-Trp-Trp-Pro)n, polyphenyalanine,polycysteine, polyarginine, a B-tag, a HSB-tag, green fluorescentprotein (GFP), influenza virus hemagglutinin (HAI), a calmodulin bindingprotein (CBP), a galactose-binding protein, a maltose binding protein(MBP), a cellulose binding domains (CBD's), dihydrofolate reductase(DHFR), glutathione-S-transferase (GST), streptococcal protein G,staphylococcal protein A, T7gene10, an avidin/streptavidin/Strep-tagcomplex, trpE, chloramphenicol acetyltransferase, lacZ(β-Galactosidase), His-patch thioredoxin, thioredoxin, a FLAG™ peptide(Sigma-Aldrich), an S-tag, or a T7-tag. See, e.g., Stevens, R. C.,Structure, 8:R177-R185 (2000). Heterologous polypeptides can alsoinclude any pre- and/or pro-sequences that facilitate the transport,translocations, processing and/or purification of a multivalentoligopeptide and/or SAg toxoid as described herein from a host cell orany useful immunogenic sequence, including but not limited to sequencesthat encode a T-cell epitope of a microbial pathogen, or otherimmunogenic proteins and/or epitopes.

In some aspects, the multivalent oligopeptide and/or SAg toxoid attachedto a heterologous polypeptide, as described herein, can include apeptide linker sequence joining sequences that comprise two or morepeptide regions. Suitable peptide linker sequences can be chosen basedon their ability to adopt a flexible, extended conformation, or asecondary structure that could interact with joined epitopes, or basedon their ability to increase overall solubility of the fusionpolypeptide, or based on their lack of electrostatic orwater-interaction effects that influence joined peptide regions.

In some aspects, the multivalent oligopeptide and/or SAg toxoid asdescribed herein, is isolated. An “isolated” polypeptide is one that hasbeen removed from its natural milieu. The term “isolated” does notconnote any particular level of purification. Recombinantly producedmultivalent oligopeptides and/or SAgs as described herein, expressed innon-native host cells is considered isolated for purposes of thedisclosure, as is the polypeptide which have been separated,fractionated, or partially or substantially purified by any suitabletechnique, including by filtration, chromatography, centrifugation, andthe like.

As provided for herein, the production of multivalent oligopeptidesand/or SAgs as described herein, can be achieved by culturing a hostcell comprising a polynucleotide that operably encodes a polypeptide ofthe disclosure, and recovering the polypeptide. Determining conditionsfor culturing such a host cell and expressing the polynucleotide aregenerally specific to the host cell and the expression system and arewithin the knowledge of one of skill in the art. Likewise, appropriatemethods for recovering the polypeptide of the disclosure are known tothose in the art, and include, but are not limited to, chromatography,filtration, precipitation, or centrifugation.

III. Polynucleotides

Also provided by this disclosure is an isolated polynucleotidecomprising a nucleic acid encoding a multivalent oligopeptide and/or SAgtoxoid as described elsewhere herein. In certain aspects, an isolatedpolynucleotide as provided herein further comprises non-coding regionssuch as promoters, operators, or transcription terminators as describedelsewhere herein. In some aspects, the disclosure is directed to thepolynucleotide as described herein, and further comprising aheterologous nucleic acid. The heterologous nucleic acid can, in someaspects, encode a heterologous polypeptide fused to the polypeptide asdescribed herein. For example, the isolated polynucleotide as describedherein can comprise additional coding regions encoding, e.g., aheterologous polypeptide fused to the polypeptide as described herein,or coding regions encoding heterologous polypeptides separate from thepolypeptide as described herein such as, but not limited to, selectablemarkers, additional immunogens, immune enhancers, and the like.

Also provided are expression constructs, vectors, and/or host cellscomprising the polynucleotides described herein. An example of anisolated polynucleotide is a recombinant polynucleotide contained in avector. In certain aspects, the vector is an expression vector. Furtherexamples of an isolated polynucleotide include recombinantpolynucleotides maintained in heterologous host cells or purified(partially or substantially) polynucleotides in solution. In certainaspects of the disclosure a polynucleotide is “recombinant.” Isolatedpolynucleotides or nucleic acids according to the disclosure furtherinclude such molecules produced synthetically. The relative degree ofpurity of a polynucleotide or polypeptide described herein is easilydetermined by well-known methods.

Also included within the scope of the disclosure are geneticallyengineered polynucleotides encoding the multivalent oligopeptides and/orSAgs as described herein. Modifications of nucleic acids encoding themultivalent oligopeptides and/or SAgs as described herein can readily beaccomplished by those skilled in the art, for example, byoligonucleotide-directed site-specific mutagenesis or de novo nucleicacid synthesis.

Some aspects disclose an isolated polynucleotide comprising a nucleicacid that encodes a multivalent oligopeptide and/or SAg toxoid asdescribed elsewhere herein, where the coding region encoding thepolypeptide has been codon-optimized. As appreciated by one of ordinaryskill in the art, various nucleic acid coding regions will encode thesame polypeptide due to the redundancy of the genetic code. Deviationsin the nucleotide sequence that comprise the codons encoding the aminoacids of any polypeptide chain allow for variations in the sequence ofthe coding region. Since each codon consists of three nucleotides, andthe nucleotides comprising DNA are restricted to four specific bases,there are 64 possible combinations of nucleotides, 61 of which encodeamino acids (the remaining three codons encode signals endingtranslation). The “genetic code” which shows which codons encode whichamino acids is reproduced herein as Table 2. As a result, many aminoacids are designated by more than one codon. For example, the aminoacids alanine and proline are coded for by four triplets, serine andarginine by six, whereas tryptophan and methionine are coded by just onetriplet. This degeneracy allows for DNA base composition to vary over awide range without altering the amino acid sequence of the polypeptidesencoded by the DNA.

TABLE 2 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S) TATTyr (Y) TGT Cys (C) TTC Phe (F) TCC Ser (S) TAC Tyr (Y) TGC TTA Leu (L)TCA Ser (S) TAA Ter TGA Ter TTG Leu (L) TCG Ser (S) TAG Ter TGG Trp (W)C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R) CTC Leu (L) CCC Pro(P) CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P) CAA Gln (Q) CGA Arg(R) CTG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R) A ATT Ile (I) ACTThr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I) ACC Thr (T) AAC Asn (N) AGCSer (S) ATA Ile (I) ACA Thr (T) AAA Lys (K) AGA Arg (R) ATG Met (M) ACGThr (T) AAG Lys (K) AGG Arg (R) G GTT Val (V) GCT Ala (A) GAT Asp (D)GGT Gly (G) GTC Val (V) GCC Ala (A) GAC Asp (D) GGC Gly (G) GTA Val (V)GCA Ala (A) GAA Glu (E) GGA Gly (G) GTG Val (V) GCG Ala (A) GAG Glu (E)GGG Gly (G)

It is to be appreciated that any polynucleotide that encodes apolypeptide in accordance with the disclosure falls within the scope ofthis disclosure, regardless of the codons used.

Many organisms display a bias for use of particular codons to code forinsertion of a particular amino acid in a growing polypeptide chain.Codon preference or codon bias, differences in codon usage betweenorganisms, is afforded by degeneracy of the genetic code, and is welldocumented among many organisms.

Different factors have been proposed to contribute to codon usagepreference, including translational selection, GC composition,strand-specific mutational bias, amino acid conservation, proteinhydropathy, transcriptional selection and even RNA stability. One factorthat determines codon usage is mutational bias that shapes genome GCcomposition. This factor is most significant in genomes with extremebase composition: species with high GC content (e.g., gram positivebacteria). Mutational bias is responsible not only for intergeneticdifference in codon usage but also for codon usage bias within the samegenome (Ermolaeva M, Curr. Issues Mol. Biol. 3(4):91-97, 2001).

Codon bias often correlates with the efficiency of translation ofmessenger RNA (mRNA), which is in turn believed to be dependent on,inter alia, the properties of the codons being translated and theavailability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization.

The present disclosure provides a polynucleotide comprising acodon-optimized coding region which encodes a multivalent oligopeptideand/or SAg toxoid as described herein. The codon usage is adapted foroptimized expression in a given prokaryotic or eukaryotic host cell. Incertain aspects the codon usage is adapted for optimized expression inE. coli.

For example, SEQ ID NO: 7 is a nucleotide sequence codon optimized forE. coli expression encoding the rTBA fusion protein:

(SEQ ID NO: 7) atgtcgacgaatgacaacatcaaagacctgctggactggtactcctcgggctcggatacgttcacgaatagcgaagtgctggcaaactcacgcggtagcatgcgtatcaaaaataccgatggtagcattagcctgatcgcttttccgtcaccgtattacagcccggcattcaccaaaggcgaaaaagtggatctgaataccaaacgcacgaaaaaatcacagcatacctcagaaggtacctacatccactttcagatcagcggcgtgaccaacaccgaaaaactgccgaccccgattgaactgccgctgaaagtgaaagttcatggcaaagattcgccgctgaaatattggccgaaatttgataaaaaacagctggcaatttcgaccctggatttcgaaattcgccaccagctgacccagatccatggtctgtaccgttcaagcgacaaaaccggcggttattggaaaatcaccatgaatgatggttcgacgtaccagagcgatctgtcgaaaaaattcgaatacaacacggaaaaaccgccgattaatatcgatgaaatcaaaaccatcgaagcggaaatcaatggcggtggcggctcgggtggtggcggtagcggtggcggcggtagtgaatcgcaaccggatccgaaaccggacgaactgcacaaatcgtccaaatttaccggtctgatggaaaatatgaaagtgctgtatgatgacaaccatgtgtcggcaattaacgtgaaaagcatcgatcagtttcgctatttcgatctgatctatagcattaaagatacgaaactgggtaattacgataacgttcgtgtggaatttaaaaacaaagatctggcggacaaatataaagacaaatacgtggacgttttcggtgcgaatgcgtattaccaatgcgcctttagcaaaaagaccaatgatatcaactcccatcagaccgacaaacgtaaaacctgcatgtacggtggtgtgaccgaacataacggtaatcagctggacaaatatcgtagcatcacggtccgtgtgtttgaagacggcaaaaacctgctgtcatttgatgttcagacgaacaaaaagaaagttacggctcaagaactggattacctgacccgccactatctggtgaaaaataaaaaactgtacgaatttaacaatagcccgtacgaaaccggctacatcaaattcattgaaaatgaaaatagcttttggtacgatatgatgccggcaccgggtgacaaatttgaccaaagcaaatacctgatgatgtacaacgataacaaaatggtcgattcaaaagacgtgaaaatcgaagtctatctgacgaccaaaaagaaaggtggcggtggttctggtggtggtggctcgggcggcggtggctcggaaaaatccgaagaaattaacgaaaaagacctgcgtaaaaaatccgaactgcagggtacggcgctgggtaatctgaaacagatttattactacaacgaaaaagccaaaaccgaaaacaaagaaagccatgatcagttccgccagcatacgatcctgttcaaaggctttttcaccgatcattcgtggtataatgacctgctggtgcgtttcgatagcaaagacattgtggataaatataaaggcaaaaaagtggatctgtatggcgcatacgctggttatcagtgtgcgggcggtacgccgaataaaacggcatgcatgtatggtggtgtgacgctgcatgacaataaccgcctgaccgaagaaaagaaagtgccgattaatctgtggctggacggtaaacagaacaccgtgccgctggaaacggtgaaaaccaataaaaagaacgtgaccgtgcaggaactggacctgcaagcacgccgttatctgcaggaaaaatataacctgtataacagcgacgtgttcgatggcaaagtgcagcgtggtctgatcgtcttccataccagcaccgaaccgagcgttaactatgacctgtttggcgcacaaggccagtactccaataccctgctgcgcatttatcgcgataacaaaaccattaactccgaaaacatgCACattgacatttacctgtacacctcgtaacatcatcaccatcattgataa taa

For example, SEQ ID NO: 8 is a nucleotide sequence codon optimized forE. coli expression encoding the rTBA225 fusion protein:

(SEQ ID NO: 8) atgtcgacgaatgacaacatcaaagacctgctggactggtactcctcgggctcggatacgttcacgaatagcgaagtgctggcaaactcacgcggtagcatgcgtatcaaaaataccgatggtagcattagcctgatcgcttttccgtcaccgtattacagcccggcattcaccaaaggcgaaaaagtggatctgaataccaaacgcacgaaaaaatcacagcatacctcagaaggtacctacatccactttcagatcagcggcgtgaccaacaccgaaaaactgccgaccccgattgaactgccgctgaaagtgaaagttcatggcaaagattcgccgctgaaatattggccgaaatttgataaaaaacagctggcaatttcgaccctggatttcgaaattcgccaccagctgacccagatccatggtctgtaccgttcaagcgacaaaaccggcggttattggaaaatcaccatgaatgatggttcgacgtaccagagcgatctgtcgaaaaaattcgaatacaacacggaaaaaccgccgattaatatcgatgaaatcaaaaccatcgaagcggaaatcaatggcggtggcggctcgggtggtggcggtagcggtggcggcggtagtgaatcgcaaccggatccgaaaccggacgaactgcacaaatcgtccaaatttaccggtctgatggaaaatatgaaagtgctgtatgatgacaaccatgtgtcggcaattaacgtgaaaagcatcgatcagtttcgctatttcgatctgatctatagcattaaagatacgaaactgggtaattacgataacgttcgtgtggaatttaaaaacaaagatctggcggacaaatataaagacaaatacgtggacgttttcggtgcgaatgcgtattaccaatgcgcctttagcaaaaagaccaatgatatcaactcccatcagaccgacaaacgtaaaacctgcatgtacggtggtgtgaccgaacataacggtaatcagctggacaaatatcgtagcatcacggtccgtgtgtttgaagacggcaaaaacctgctgtcatttgatgttcagacgaacaaaaagaaagttacggctcaagaactggattacctgacccgccactatctggtgaaaaataaaaaactgtacgaatttaacaatagcccgtacgaaaccggctacatcaaattcattgaaaatgaaaatagcttttggtacgatatgatgccggcaccgggtgacaaatttgaccaaagcaaatacctgatgatgtacaacgataacaaaatggtcgattcaaaagacgtgaaaatcgaagtctatctgacgaccaaaaagaaaggtggcggtggttctggtggtggtggctcgggcggcggtggctcggaaaaatccgaagaaattaacgaaaaagacctgcgtaaaaaatccgaactgcagggtacggcgctgggtaatctgaaacagatttattactacaacgaaaaagccaaaaccgaaaacaaagaaagccatgatcagttccgccagcatacgatcctgttcaaaggctttttcaccgatcattcgtggtataatgacctgctggtgcgtttcgatagcaaagacattgtggataaatataaaggcaaaaaagtggatctgtatggcgcatacgctggttatcagtgtgcgggcggtacgccgaataaaacggcatgcatgtatggtggtgtgacgctgcatgacaataaccgcctgaccgaagaaaagaaagtgccgattaatctgtggctggacggtaaacagaacaccgtgccgctggaaacggtgaaaaccaataaaaagaacgtgaccgtgcaggaactggacctgcaagcacgccgttatctgcaggaaaaatataacctgtataacagcgacgtgttcgatggcaaagtgcagcgtggtctgatcgtcttccataccagcaccgaaccgagcgttaactatgacctgtttggcgcacaaggccagtactccaataccctgctgcgcatttatcgcgataacaaaaccattaactccgaaaacatgGCCattgacatttacctgtacacctcgtaacatcatcaccatcattgataa taa

Codon-optimized polynucleotides are prepared by incorporating codonspreferred for use in the genes of a given species into the DNA sequence.Also provided are polynucleotide expression constructs, vectors, hostcells comprising polynucleotides comprising codon-optimized codingregions which encode a multivalent oligopeptide and/or SAg toxoid asdescribed herein.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, it is possible to calculate therelative frequencies of codon usage. Codon usage tables are readilyavailable, for example, at the “Codon Usage Database” available athttp://www.kazusa.or.jp/codon/(visited Oct. 12, 2011), and these tablescan be adapted in a number of ways. (Nakamura, Y., et al., “Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292, 2000).

By utilizing available tables, one of ordinary skill in the art canapply the frequencies to any given polypeptide sequence, and produce anucleic acid fragment of a codon-optimized coding region which encodes adesired polypeptide, but which uses codons optimal for a given species.A number of options are available for synthesizing codon optimizedcoding regions designed by any of the methods described above, usingstandard and routine molecular biological manipulations well known tothose of ordinary skill in the art. In addition, gene synthesis isreadily available commercially.

IV. Vectors and Expression Systems

Further provided is a vector comprising a polynucleotide as providedherein. The term “vector,” as used herein, refers to e.g., any of anumber of nucleic acids into which a desired sequence can be inserted,e.g., by restriction and ligation, for transport between differentgenetic environments or for expression in a host cell. Nucleic acidvectors can be DNA or RNA. Vectors include, but are not limited to,plasmids, phage, phagemids, bacterial genomes, and virus genomes. Acloning vector is one which is able to replicate in a host cell, andwhich is further characterized by one or more endonuclease restrictionsites at which the vector can be cut in a determinable fashion and intowhich a desired DNA sequence can be ligated such that the newrecombinant vector retains its ability to replicate in the host cell. Inthe case of plasmids, replication of the desired sequence can occur manytimes as the plasmid increases in copy number within the host bacteriumor just a single time per host before the host reproduces by mitosis. Inthe case of phage, replication can occur actively during a lytic phaseor passively during a lysogenic phase. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced.Other vectors are integrated into the genome of a host cell uponintroduction into the host cell, and thereby are replicated along withthe host genome.

Any of a wide variety of suitable cloning vectors are known in the artand commercially available which can be used with appropriate hosts. Asused herein, the term “plasmid” refers to a circular, double-strandedconstruct made up of genetic material (i.e., nucleic acids), in whichthe genetic material is extrachromosomal and in some instances,replicates autonomously. A polynucleotide described herein can be in acircular or linearized plasmid or in any other sort of vector.Procedures for inserting a nucleotide sequence into a vector, e.g., anexpression vector, and transforming or transfecting into an appropriatehost cell and cultivating under conditions suitable for expression aregenerally known in the art.

The disclosure further provides a vector comprising a nucleic acidsequence encoding a multivalent oligopeptide and/or SAg toxoid asdescribed elsewhere herein. In certain aspects the vector is anexpression vector capable of expressing the multivalent oligopeptideand/or SAg toxoid as described herein in a suitable host cell. The term“expression vector” refers to a vector that is capable of expressing thepolypeptide described herein, i.e., the vector sequence contains theregulatory sequences regulating transcription and translation of apolypeptide, including, but not limited to promoters, operators,transcription termination sites, ribosome binding sites, and the like.The term “expression” refers to the biological production of a productencoded by a coding sequence. In most cases a DNA sequence, includingthe coding sequence, is transcribed to form a messenger-RNA (mRNA). Themessenger-RNA is then translated to form a polypeptide product which hasa relevant biological activity. Also, the process of expression caninvolve further processing steps to the RNA product of transcription,such as splicing to remove introns, and/or post-translational processingof a polypeptide product.

Vector-host systems include, but are not limited to, systems such asbacterial, mammalian, yeast, insect or plant cell systems, either invivo, e.g., in an animal or in vitro, e.g., in bacteria or in cellcultures. The selection of an appropriate host is deemed to be withinthe scope of those skilled in the art from the teachings herein. Incertain aspects, the host cell is a bacterium, and insect cell, amammalian cell, or a plant cell. In certain aspects, the bacterium is E.coli.

Host cells are genetically engineered (infected, transduced,transformed, or transfected) with vectors of the disclosure. Thus, oneaspect of the disclosure is directed to a host cell comprising a vectorwhich contains the polynucleotide as describe herein. The engineeredhost cell can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants oramplifying the polynucleotides. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan. The term “transfect,” as used herein, refers to anyprocedure whereby eukaryotic cells are induced to accept and incorporateinto their genome isolated DNA, including but not limited to DNA in theform of a plasmid. The term “transform,” as used herein, refers to anyprocedure whereby bacterial cells are induced to accept and incorporateinto their genome isolated DNA, including but not limited to DNA in theform of a plasmid.

Bacterial host-expression vector systems include, but are not limitedto, a prokaryote (e.g., E. coli), transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA. In some aspects, theplasmids used with E. coli use the T7 promoter-driven system regulatedby the LacI protein via IPTG induction. A large number of suitablevectors are known to those of skill in the art, and are commerciallyavailable. The following bacterial vectors are provided by way ofexample: pET (Novagen), pET28, pBAD, pTrcHIS, pBR322, pQE70, pQE60,pQE-9 (Qiagen), phagescript, psiX174, pBluescript SK, pbsks, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK243-3,pDR540, pBR322, pPS10, RSF1010, pRIT5 (Pharmacia); pCR (Invitrogen);pLex (Invitrogen), and pUC plasmid derivatives.

A suitable expression vector contains regulatory sequences that can beoperably joined to an inserted nucleotide sequence encoding themultivalent oligopeptide and/or SAg toxoid as described herein. As usedherein, the term “regulatory sequences” means nucleotide sequences whichare necessary for or conducive to the transcription of an insertedsequence encoding a multivalent oligopeptide and/or SAg toxoid asdescribed herein by a host cell and/or which are necessary for orconducive to the translation by a host cell of the resulting transcriptinto the desired multivalent oligopeptide and/or SAg toxoid. Regulatorysequences include, but are not limited to, 5′ sequences such asoperators, promoters and ribosome binding sequences, and 3′ sequencessuch as polyadenylation signals or transcription terminators. Regulatorysequences can also include enhancer sequences or upstream activatorsequences.

Generally, bacterial vectors will include origins of replication andselectable markers, e.g., the ampicillin, tetracycline, kanamycin,resistance genes of E. coli, permitting transformation of the host celland a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Suitable promotersinclude, but are not limited to, the T7 promoter, lambda (k) promoter,T5 promoter, and lac promoter, or promoters derived from operonsencoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK),acid phosphatase, or heat shock proteins, or inducible promoters likecadmium (pcad), and beta-lactamase (pbla).

Once an expression vector is selected, the polynucleotide as describedherein can be cloned downstream of the promoter, for example, in apolylinker region. The vector is transformed into an appropriatebacterial strain, and DNA is prepared using standard techniques. Theorientation and DNA sequence of the polynucleotide as well as all otherelements included in the vector, are confirmed using restrictionmapping, DNA sequence analysis, and/or PCR analysis. Bacterial cellsharboring the correct plasmid can be stored as cell banks.

V. Immunogenic and Pharmaceutical Compositions

Further disclosed are compositions, e.g., immunogenic or pharmaceuticalcompositions that contain an effective amount of the multivalentoligopeptide and/or SAg toxoid as described herein, or a polynucleotideencoding the polypeptide of the disclosure. Compositions as describedherein can further comprise additional immunogenic components, e.g., asa multivalent vaccine, as well as carriers, excipients or adjuvants.

Compositions as provided herein can be formulated according to knownmethods. Suitable preparation methods are described, for example, inRemington's Pharmaceutical Sciences, 19th Edition, A. R. Gennaro, ed.,Mack Publishing Co., Easton, Pa. (1995), which is incorporated herein byreference in its entirety. Composition can be in a variety of forms,including, but not limited to an aqueous solution, an emulsion, a gel, asuspension, lyophilized form, or any other form known in the art. Inaddition, the composition can contain pharmaceutically acceptableadditives including, for example, diluents, binders, stabilizers, andpreservatives. Once formulated, compositions of the disclosure can beadministered directly to the subject. The subjects to be treated can beanimals; in particular, human subjects can be treated.

Carriers that can be used with compositions of the disclosure are wellknown in the art, and include, without limitation, e.g., thyroglobulin,albumins such as human serum albumin, tetanus toxoid, and polyaminoacids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitisB virus core protein, and the like. A variety of aqueous carriers can beused, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronicacid and the like. Compositions can be sterilized by conventional, wellknown sterilization techniques, or can be sterile filtered. A resultingcomposition can be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. Compositions can contain pharmaceutically acceptableauxiliary substances as to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents and the like, for example, sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamineoleate, etc.

Certain compositions as provided herein further include one or moreadjuvants, a substance added to an immunogenic composition to, forexample, enhance, sustain, localize, or modulate an immune response toan immunogen. The term “adjuvant” refers to any material having theability to (1) alter or increase the immune response to a particularantigen or (2) increase or aid an effect of a pharmacological agent. Anycompound which can increase the expression, antigenicity orimmunogenicity of the polypeptide is a potential adjuvant. The term“immunogenic carrier” as used herein refers to a first moiety, e.g., apolypeptide or fragment, variant, or derivative thereof which enhancesthe immunogenicity of a second polypeptide or fragment, variant, orderivative thereof.

A great variety of materials have been shown to have adjuvant activitythrough a variety of mechanisms. For example, an increase in humoralimmunity is typically manifested by a significant increase in the titerof antibodies raised to the antigen, and an increase in T-cell activityis typically manifested in increased cell proliferation, or cellularcytotoxicity, or cytokine secretion. An adjuvant can also alter ormodulate an immune response, for example, by changing a primarilyhumoral or Th₂ response into a primarily cellular, or Th₁ response.Immune responses to a given antigen can be tested by variousimmunoassays well known to those of ordinary skill in the art, and/ordescribed elsewhere herein.

A wide number of adjuvants are familiar to persons of ordinary skill inthe art, and are described in numerous references. Adjuvants which canbe used in compositions described herein include, but are not limitedto: inert carriers, such as alum, bentonite, latex, and acrylicparticles; incomplete Freund's adjuvant, complete Freund's adjuvant;aluminum-based salts such as aluminum hydroxide; Alhydrogel (Al(OH₃));aluminum phosphate (A1PO₄); calcium-based salts; silica; any TLRbiological ligand(s); IDC-1001 (also known as GLA-SE; glucopyranosyllipid adjuvant stable emulsion) (Coler et al., PLoS One, 2010. 5(10): p.e13677; Coler et al., PLoS One, 2011. 6(1): p. e16333); CpG (Mullen etal., PLoS One, 2008. 3(8): p. e2940), or any combination thereof. Incertain aspects, the adjuvant comprises Alhydrogel. The amount ofadjuvant, how it is formulated, and how it is administered allparameters which are well within the purview of a person of ordinaryskill in the art.

In some aspects, a composition of the disclosure further comprises aliposome or other particulate carrier, which can serve, e.g., tostabilize a formulation, to target the formulation to a particulartissue, such as lymphoid tissue, or to increase the half-life of thepolypeptide composition. Such particulate carriers include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers, iscoms, and the like. In thesepreparations, the polypeptide described herein can be incorporated aspart of a liposome or other particle, or can be delivered in conjunctionwith a liposome. Liposomes for use in accordance with the disclosure canbe formed from standard vesicle-forming lipids, which generally includeneutral and negatively charged phospholipids and a sterol, such ascholesterol. A composition comprising a liposome or other particulatesuspension as well as the polypeptide as described herein can beadministered intravenously, locally, topically, etc. in a dose whichvaries according to, inter alia, the manner of administration, thepolypeptide being delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers can be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, the polypeptide as described herein, often at aconcentration of 25%-75%.

For aerosol or mucosal administration, the polypeptide as describedherein can be supplied in finely divided form, optionally along with asurfactant and, propellant and/or a mucoadhesive, e.g., chitosan. Incertain aspects, the surfactant is pharmaceutically acceptable, and insome aspects soluble in the propellant. Representative of such agentsare the esters or partial esters of fatty acids containing from 6 to 22carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic,linoleic, linolenic, olesteric and oleic acids with an aliphaticpolyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixedor natural glycerides can be employed. The surfactant can constitute0.1%-20% by weight of the composition, in some aspects 0.25-5% byweight. The balance of the composition is ordinarily propellant,although an atomizer can be used in which no propellant is necessary andother percentages are adjusted accordingly. In some aspects, theimmunogenic polypeptides can be incorporated within an aerodynamicallylight particle, such as those particles described in U.S. Pat. No.6,942,868 or U.S. Pat. Pub. No. 2005/0008633. A carrier can also beincluded, e.g., lecithin for intranasal delivery.

The disclosure is also directed to a method of producing the compositionaccording to the disclosure. In some aspects, the method of producingthe composition comprises (a) isolating a polypeptide according to thedisclosure; and (b) adding an adjuvant, carrier and/or excipient to theisolated polypeptide. Some aspects disclose further combining thepolypeptide with other staphylococcal antigens.

Some aspects include a multivalent vaccine. A multivalent vaccine of thepresent disclosure can include a multivalent oligopeptide and/or SAgtoxoid as described herein, or a polynucleotide encoding a multivalentoligopeptide and/or SAg toxoid, and one or more additional immunogeniccomponents. Such components can be additional immunogens of the sameinfectious agent, e.g., S. aureus, or from other staphylococci, or canbe immunogens derived from other infectious agents which can beeffectively, conveniently, or economically administered together. Incertain aspects, the multivalent oligopeptide and/or SAg toxoid asdescribed herein, can be combined with other toxins or other virulentcomponent-based vaccines to make a broad toxin-based multivalent vaccinecapable of targeting multiple bacterial virulence determinants. In otheraspects, the multivalent oligopeptide and/or SAg toxoid as describedherein can be fused to other immunogenic, biologically significant, orprotective epitope containing polypeptides to generate a multivalentvaccine in a single chain and induce an immune response against multipleantigens. In yet another aspect, the multivalent oligopeptide and/or SAgtoxoid as described herein, can be fused to one or more T cell epitopesto induce T cell immunity.

VI. Methods of Treatment/Prevention and Regimens

Also provided is a method of treating or preventing Staphylococcusinfection, e.g., S. aureus infection or treating or preventing a diseasecaused by Staphylococcus, e.g, S. aureus in a subject, comprisingadministering to a subject in need thereof a composition as describedherein comprising a multivalent oligopeptide and/or SAg toxoid asdescribed herein, or polynucleotides, vectors, or host cells encodingsame. In certain aspects, the subject is an animal, e.g., a vertebrate,e.g., a mammal, e.g., a human. Some aspects include a method of inducingan immune response against a S. aureus strain, comprising administeringto a subject in need of said immune response an effective amount of acomposition comprising a multivalent oligopeptide and/or SAg toxoid asdescribed herein, or polynucleotides, vectors, or host cells encodingsame.

In some aspects, a subject is administered a composition comprising amultivalent oligopeptide and/or SAg toxoid as described herein, orpolynucleotides, vectors, or host cells encoding same prophylactically,e.g., as a prophylactic vaccine, to establish or enhance immunity toStaphylococcus, e.g., S. aureus, in a healthy animal prior to potentialor actual exposure to Staphylococcus, e.g., S. aureus or contraction ofaStaphylococcus-related symptom, thus preventing disease, alleviatingsymptoms, reducing symptoms, or reducing the severity of diseasesymptoms. In one aspect the disease is a respiratory disease, e.g.,pneumonia. Other diseases or conditions to be treated or preventedinclude, but are not limited to, bacteremia, sepsis, skin infections,wound infections, endocarditis, bone and joint infections,osteomyelitis, and/or meningitis. One or more compositions,polypeptides, polynucleotides, vectors, or host cells as describedherein can also be used to treat a subject already exposed toStaphylococcus, e.g., S. aureus, or already suffering from aStaphylococcus related symptom to further stimulate the immune system ofthe animal, thus reducing or eliminating the symptoms associated withthat exposure. As defined herein, “treatment of an animal” refers to theuse of one or more compositions, polypeptides, polynucleotides, vectors,or host cells of the disclosure to prevent, cure, retard, or reduce theseverity of S. aureus symptoms in an animal, and/or result in noworsening of S. aureus symptoms over a specified period of time. It isnot required that any composition, polypeptide, polynucleotide, avector, or a host cell as described herein provides total protectionagainst a staphylococcal infection or totally cure or eliminate allStaphylococcus related symptoms.

As used herein, “a subject in need of therapeutic and/or preventativeimmunity” refers to a subject in which it is desirable to treat, i.e.,to prevent, cure, retard, or reduce the severity of Staphylococcusrelated symptoms, or result in no worsening of Staphylococcus relatedsymptoms over a specified period of time. As used herein, “a subject inneed of the immune response” refers to a subject for which an immuneresponse(s) against a S. Staphylococcus related disease is desired.

Treatment with pharmaceutical compositions comprising an immunogeniccomposition, polypeptide or polynucleotide as described herein can occurseparately or in conjunction with other treatments, as appropriate.

In therapeutic applications, a composition, polypeptide orpolynucleotide of the disclosure is administered to a patient in anamount sufficient to elicit an effective innate, humoral and/or cellularresponse to the multivalent oligopeptide and/or SAg toxoid to cure or atleast partially arrest symptoms or complications.

An amount adequate to accomplish this is defined as “therapeuticallyeffective dose” or “unit dose.” Amounts effective for this use willdepend on, e.g., the polypeptide or polynucleotide composition, themanner of administration, the stage and severity of the disease beingtreated, the weight and general state of health of the patient, and thejudgment of the prescribing physician. In some aspects, a priming doseis followed by a boosting dose over a period of time.

In some aspects, generally for humans, an initial immunization (that isfor therapeutic or prophylactic administration) is administered followedby boosting dosages in the same dose range pursuant to a boostingregimen over weeks to months depending upon the patient's response andcondition by measuring the antibody or T lymphocyte response in thepatient's blood.

Polypeptides and compositions as described herein can generally beemployed in serious disease states, that is, life-threatening orpotentially life threatening situations. In such cases, in view of theminimization of extraneous substances and the relative nontoxic natureof the polypeptides, it is possible and can be felt desirable by thetreating physician to administer substantial excesses of thesepolypeptide compositions.

For therapeutic use, administration can begin at the first sign of S.aureus infection or risk factors. In certain aspects, the initial doseis followed by boosting doses until, e.g., symptoms are substantiallyabated and for a period thereafter. In frequent infection, loading dosesfollowed by boosting doses can be indicated.

In certain aspects, the composition as described herein is delivered toa subject by methods described herein, thereby achieving an effectiveimmune response, and/or an effective therapeutic or preventative immuneresponse. Any mode of administration can be used so long as the moderesults in the delivery and/or expression of the desired polypeptide inthe desired tissue, in an amount sufficient to generate an immuneresponse to Staphylococcus, e.g., S. aureus, and/or to generate aprophylactically or therapeutically effective immune response toStaphylococcus, e.g., to S. aureus, in an animal in need of suchresponse. According to the disclosed methods, a composition describedherein can be administered by mucosal delivery, transdermal delivery,subcutaneous injection, intravenous injection, oral administration,pulmonary administration, intramuscular (i.m.) administration, or viaintraperitoneal injection. Other suitable routes of administrationinclude, but not limited to intratracheal, transdermal, intraocular,intranasal, inhalation, intracavity, intraductal (e.g., into thepancreas) and intraparenchymal (i.e., into any tissue) administration.Transdermal delivery includes, but not limited to intradermal (e.g.,into the dermis or epidermis), transdermal (e.g., percutaneous) andtransmucosal administration (i.e., into or through skin or mucosaltissue). Intracavity administration includes, but not limited toadministration into oral, vaginal, rectal, nasal, peritoneal, orintestinal cavities as well as, intrathecal (i.e., into spinal canal),intraventricular (i.e., into the brain ventricles or the heartventricles), intra-arterial (i.e., into the heart atrium) and subarachnoidal (i.e., into the sub arachnoid spaces of the brain)administration.

Any mode of administration can be used so long as the mode results inthe delivery and/or expression of the desired polypeptide in an amountsufficient to generate an immune response to Staphylococcus, e.g., S.aureus, and/or to generate a prophylactically or therapeuticallyeffective immune response to Staphylococcus, e.g., S. aureus, in ananimal in need of such response. Administration as described herein canbe by e.g., needle injection, or other delivery or devices known in theart.

In some aspects, a composition comprising a multivalent oligopeptideand/or SAg toxoid as described herein, or polynucleotides, vectors, orhost cells encoding same, stimulate an antibody response or acell-mediated immune response sufficient for protection of an animalagainst Staphylococcus, e.g., S. aureus infection. In other aspects, acomposition comprising a multivalent oligopeptide and/or SAg toxoid asdescribed herein, or polynucleotides, vectors, or host cells encodingsame, stimulate both a humoral and a cell-mediated response, thecombination of which is sufficient for protection of an animal againstStaphylococcus, e.g., S. aureus infection. In some aspects, acomposition comprising a multivalent oligopeptide and/or SAg toxoid asdescribed herein, or polynucleotides, vectors, or host cells encodingsame, further stimulates an innate, an antibody, and/or a cellularimmune response.

In some aspects, a composition comprising a multivalent oligopeptideand/or SAg toxoid as described herein, or polynucleotides, vectors, orhost cells encoding same, can induce antibody responses to S. aureus. Incertain aspects, components that induce T cell responses (e.g., T cellepitopes) are combined with components such as the polypeptides asdescribed herein that primarily induce an antibody response.

Further disclosed is a method for generating, enhancing, or modulating aprotective and/or therapeutic immune response to S. aureus infection ina subject, comprising administering to a subject in need of therapeuticand/or preventative immunity one or more of the compositions asdescribed herein.

The compositions as described herein can be administered to an animal atany time during the lifecycle of the animal to which it is beingadministered. In humans, administration of the composition as describedherein can, and often advantageously occurs while other vaccines arebeing administered, e.g., as a multivalent vaccine as describedelsewhere herein.

Furthermore, the composition as described herein can be used in anydesired immunization or administration regimen; e.g., in a singleadministration or alternatively as part of periodic vaccination regimessuch as annual vaccinations, or as in a prime-boost regime in whichcomposition or polypeptide or polynucleotide of the disclosure isadministered either before or after the administration of the same or ofa different polypeptide or polynucleotide. Recent studies have indicatedthat a prime-boost protocol is often a suitable method of administeringvaccines. In a prime-boost protocol, one or more compositions asdescribed herein can be utilized in a “prime boost” regimen. An exampleof a “prime boost” regimen can be found in Yang, Z. et al. J. Virol.77:799-803, 2002, which is incorporated herein by reference in itsentirety.

Infections to be treated include, but are not limited to a localized orsystemic infection of skin, soft tissue, blood, or an organ or anauto-immune disease. Specific diseases or conditions to be treated orprevented include, but are not limited to, respiratory diseases, e.g.,pneumonia, sepsis, skin infections, wound infections, endocarditis, boneand joint infections, osteomyelitis, and/or meningitis.

A number of animal models for S. aureus infection are known in the art,and can be used with the methods disclosed herein without undueexperimentation. For example, a hamster model of methicillin-resistantStaphylococcus aureus (MRSA) pneumonia has been described for thetesting of antimicrobials. (Verghese A. et al., Chemotherapy. 34:497-503(1988), Kephart P A. et al. J Antimicrob Chemother. 21:33-9, (1988)).Further, a model of S. aureus-induced pneumonia in adult,immunocompetent C57BL/6J mice is described, which closely mimics theclinical and pathological features of pneumonia in human patients.(Bubeck-Wardenburg J. et al., Infect Immun. 75:1040-4 (2007)).Additionally, virulence has been tested in a rat model of S. aureuspneumonia as described in McElroy et al. (McElroy M C. et al., InfectImmun. 67:5541-4 (1999)). Finally, a standardized and reproducible modelof MRSA-induced septic pneumonia to evaluate new therapies wasestablished in sheep. (Enkhbaatar P. et al., Shock. 29(5):642-9 (2008)).

The practice of the disclosure will employ, unless otherwise indicated,conventional techniques of cell biology, cell culture, molecularbiology, transgenic biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., Sambrook et al., ed., Cold Spring HarborLaboratory Press: (1989); Molecular Cloning: A Laboratory Manual,Sambrook et al., ed., Cold Springs Harbor Laboratory, New York (1992),DNA Cloning, D. N. Glover ed., Volumes I and II (1985); OligonucleotideSynthesis, M. J. Gait ed., (1984); Mullis et al. U.S. Pat. No.4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds.(1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds.(1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc.,(1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors ForMammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al.eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer andWalker, eds., Academic Press, London (1987); Handbook Of ExperimentalImmunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., (1986);Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein, J., Immunology: The Science of Self-Nonself Discrimination, JohnWiley & Sons, New York (1982); Roitt, I., Brostoff, J. and Male D.,Immunology, 6^(th) ed. London: Mosby (2001); Abbas A., Abul, A. andLichtman, A., Cellular and Molecular Immunology, Ed. 5, Elsevier HealthSciences Division (2005); and Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Press (1988).

EXAMPLES

The breadth and scope of the present disclosure should not be limited byany of the above-described exemplary aspects or embodiments, but shouldbe defined only in accordance with the following claims and theirequivalents.

Example 1: rTBA225 Triple Fusion of Staphylococcal Superantigen Toxoids

While the safety of rSEB has been extensively evaluated including aphase I clinical trial, the safety of rSEA and rTSST-1 have not beenextensively studied. In addition, to evaluate whether the fusion of thethree superantigen toxoids exacerbate residual superantigenic activity,the response of PBMC from healthy human donors to rTBA using IFN7release as readout for superantigenic activity was evaluated (“PBMCstimulation assay”).

For the PBMC stimulation assay, PBMC were incubated in culture medium inindividual wells of 96 well plates with various concentration of wildtype TSST-1 or toxoids such as rTBA at 37° C. and 5% CO₂ in a humidifiedincubator. After 48 hours of culture, the plates were centrifuged for 5minutes and supernatants removed. The IFNγ concentration in each wellwas measured using an ELISA kit from R&D Systems according tomanufacturer's instructions. The concentration of induced IFNγ wasplotted against the concentration of the toxin or toxoid to determineEC₅₀ (50 percent effective concentration) for each agent.

Three donors characterized as low, medium, and high responders wereused. As shown in FIG. 4, at high concentrations, rTBA exhibited lowlevel of IFN-γ induction in the low responder, medium level in mediumresponder and high levels in high responders, although these responseswere much lower than the responses of the same donor to wild-typesuperantigens.

These experiments suggested that rTBA retains some residualsuperantigenic activity. Further analysis indicated that this activityis due to residual activity of rSEAL48R/D70R/Y92A, whilerTSSTL30R/D27A/I46A was completely inactive. Therefore, an additionalmutation was introduced into the rSEA portion of rTBA. A previous reportsuggested that mutation of H225 (SEA-H225A) binding site for MHC classII reduced the ability of SEA to stimulate T cells (Hudson et al., 1995,Journal/J Exp Med, 182(3):711-720; Kozono et al., 1995,Journal/Immunity, 3(2):187-196). A mutation was introduced at positionH225A into WT SEA, rSEAL48R/D70R/Y92A as well as rTBA. The new mutantsare referred to herein as SEAH225A (SEQ ID NO: 11), rSEAVax225 (also asSEA_(L48R/D70R/Y92A/H225A)) (SEQ ID NO: 4), and rTBA225 (SEQ ID NO: 6),respectively, and were tested in the PBMC stimulation assay.Introduction of the single H225A mutation into wild-type SEA (SEAH225A)attenuated the toxin but left significant levels of residual toxicity(FIG. 4). The combination of H225A and the L48R/D70R/Y92A mutations(rSEAVax225), however, was completely inactive on low and mediumresponder cells and only marginally active on high responder cells atvery high concentration (FIG. 4). Similarly, rTBA showed residualtoxicity while rTBA225 was completely attenuated, even more so thanrSEAVax225. These data indicate that a combination of these fourmutations was required for full attenuation of rTBA and rSEAVax (FIG.4).

Example 2: Method for Production and Tag-Free Purification of FusionProtein of Superantigen Mutants

The genes encoding the fusion of toxoids rTBA (SEQ ID NO: 5) and rTBA225(SEQ ID NO: 6) were codon optimized, synthesized, cloned into the pET24a(+) expression vector, and transformed into BL21(DE3) E. coli cells.Overnight cultures were expanded in Luria Broth containing kanamycinuntil a mid-log phase culture (˜0.5 OD at 600 nm), at which point thecells were chilled to ˜25° C. and induced with 0.3 mM IPTG, followed byovernight culture at 25° C. The next day, the bacterial cells wereharvested, weighed, and resuspended in cell lysis buffer (20 mM Tris pH8.0, 50 mM NaCl, 1 mM EDTA, 0.1% Triton X-100). Lysozyme was added (1mg/mL), and the cells were incubated at 37° C. for 30 minutes. Thepartially lysed cells were sonicated. Bacterial cell lysis was confirmedspectrophotometrically. The cell lysate was adjusted to 0.5 M NaCl, andthe nucleic acid was precipitated by the addition of polyethyleneimine(PEI) under constant mixing. The PEI pellet was removed bycentrifugation, and the supernatant containing the toxoid was subjectedto ammonium sulfate ((NH₄)₂SO₄) precipitation. The (NH₄)₂SO₄ pellet wasrecovered by centrifugation and stored at −80° C.

As shown in FIG. 2A, the following chromatography steps were performed.The (NH₄)₂SO₄ pellets were resuspended and desalted into the capturecolumn equilibration buffer, clarified, and subjected to chromatographyover a Poros 50 HS column. The column was equilibrated, loaded, washedand eluted using a 40-column volume (CV) gradient from 25 to 1,000 mMNaCl in phosphate buffer at pH 6.5. The column fractions were analyzedby SDS-PAGE to determine the toxoid containing fractions. The pooledmaterial was dialyzed into the next column equilibration buffer andsubjected to chromatography over a BioRad Ceramic Hydroxyapatite (HTP)Type I column. The column was equilibrated, loaded, washed and elutedusing a 40 CV gradient of 50-1,000 mM NaCl in a phosphate buffer at pH6.8. The fractions were analyzed by SDS-PAGE to detect the toxoid (FIG.2B). The pooled HTP fractions were dialyzed into the appropriate storagebuffer, filter sterilized, aliquoted and frozen at −80° C.

Example 3: Immunogenicity of the Fusion Construct rTBA

Groups of 5 BALB/c mice were immunized, 3 times with 14 day interval,with either rTBA or a cocktail of the three toxoids along with SigmaAdjuvant System (SAS) adjuvant. Day 35 sera from these mice were testedfor total antibody ELISA and toxin neutralization (TNA) titers.

Peripheral blood mononuclear cells(PBMC) were isolated from heparinizedblood of healthy human donors by Ficoll gradient centrifugation.Isolated PBMCs were re-suspended in RPMI 1640 with 5% fetal bovine serum(FBS), cells were washed, enumerated by Trypan blue exclusion andadjusted to 2×10⁶ cells/ml. 75 μl of this cell suspension (1.5×10⁵cells) with a viability of >95% was added to duplicate wells of 96-wellflat-bottom plates containing 37.5 μl of semi-log diluted sera fromvaccinated animals mixed with a fixed concentration of the superantigen.Wells containing medium with toxin only were used as controls. Thecultures were incubated at 37° C. in an atmosphere of 5% CO₂₋₉₅% air for48 hours. Cells were centrifuged at 1600×g for 10 minutes, culturesupernatants were harvested and IFNγ production was assessed by ELISA(R&D Systems, Minneapolis, Minn.) following the manufacturers' protocol.Plates were read at 450 nm using the VersaMax plate reader and data wastransferred and analyzed in Microsoft Office Excel 2007. Cellsstimulated with toxin in the absence of a neutralizing antibodies servedas positive control and was considered as 0% IFNγ inhibition.Accordingly, inhibition of IFNγ production in the presence of immunesera was calculated as the difference between positive control andsample. IC₅₀ values for the neutralizing agents (human monoclonalantibodies) were determined using a 4-parameter logistic model (equation205, XLFit v5.2).

rTBA induced much higher titers of total IgG binding to SEB and TSST-1as well as higher toxin neutralization (TNA) titers as compared to thecocktail of the three toxoids (FIG. 3A). Thus, the fusion of the threetoxoids into one molecule not only simplifies the vaccine, but alsoenhances the immunogenicity.

The immunogenicity of rTBA formulated in Alhydrogel or CpG was alsocompared. As shown in FIG. 3B, both adjuvants induced very high andbalanced titers against all three toxins and the magnitude of antibodyresponse was higher than those achieved with SAS adjuvant. The twoadjuvants were equivalent with respect to induction of neutralizingantibodies against SEB and TSST-1 while Alhydrogel induced strongerneutralizing response against SEA.

Example 4: Immunogenicity of Fusion Construct rTBA225

The immunogenicity of rTBA225 was tested in Balb/c mice in comparison torTBA to determine whether the additional mutation impacted theimmunogenicity. Mice were immunized three times with 20 μg either of SAgcocktail (equimolar amounts of each individual toxoid), rTBA, or rTBA225along with Alhydrogel. After the third immunization, mouse sera weretested for binding and neutralization titers by ELISAs and toxinneutralization assay (TNA) for the antigens SEA, SEB and TSST-1. Asshown in FIG. 5, mice vaccinated with the fusion constructs had a strongtotal antibody (FIG. 5A) and neutralizing antibody response (FIG. 5B) toall three superantigens. These data show that addition of the mutationdid not reduce the immunogenicity of the fusion vaccine. Furthermore,the fusion protein rTBA225 is able to induce neutralizing activitytowards superantigens that are not included in the antigen as shown inFIG. 5C.

It was observed that the SAg toxoid cocktail formulated in Alhydrogelwas unable to induce any antibody response to TSST-1, while in sharpcontrast, the fusion proteins rTBA and rTBA225 induced strong TSST-1response (FIG. 5A left panel). These data show that fusion of TSST-1 wasnecessary for inducing strong immune response when formulated withAlhydrogel. The binding data indicated that this is due to inability ofTSST-1 alone to adsorb Alhydrogel, while as a fusion protein the antigenadsorbs the Alhydrogel and therefore can induce strong antibody response(FIG. 6).

Example 5: Protective Efficacy of rTBA225 Vaccine Against ToxinChallenge with SEA, SEB, and TSST-1

The protective efficacy of rTBA225 against SAg toxin challenge wasevaluated by immunizing Balb/c mice with 20 μg of rTBA225 thrice alongwith Alhydrogel as the adjuvant followed by challenge with anintraperitoneal lethal dose of SEA (10 μg/mouse), SEB (3.315 μg/mouse)or TSST-1 (10 μg/mouse) potentiated by 40 μg/mouse LPS. Weights andhealth scores of the mice were monitored for five days after thechallenge. As shown in FIG. 7, immunization with rTBA225 provided 100%protection to SEB and TSST-1 challenge and 90% protection to SEAchallenge. These data demonstrate the protective efficacy of rTBA225against challenge with the respective toxins.

1-58. (canceled)
 59. An isolated polynucleotide comprising a nucleicacid that encodes an attenuated Staphylococcus aureus-derivedsuperantigen (SAg) SEA toxoid comprising an amino acid sequence that isat least 95% identical to SEQ ID NO: 4, wherein the toxoid comprises thefour mutations relative to wild-type SEA corresponding to the L48R,D70R, Y92A, and H225A mutations and wherein the toxoid has decreasedsuperantigenic activity and/or is less virulent than a SEA toxoidcomprising SEQ ID NO:
 3. 60. The isolated polynucleotide of claim 59,wherein the attenuated SEA toxoid comprises an amino acid sequence atleast 97% identical to SEQ ID NO:
 4. 61. The isolated polynucleotide ofclaim 59, wherein the attenuated SEA toxoid comprises SEQ ID NO:
 4. 62.The isolated polynucleotide of claim 59, comprising the nucleotidesequence SEQ ID NO:
 8. 63. The isolated polynucleotide of claim 59,further comprising a heterologous nucleic acid.
 64. The isolatedpolynucleotide of claim 59, wherein the heterologous nucleic acidcomprises a promoter operably associated with the nucleic acid encodingthe oligopeptide.
 65. A vector comprising the isolated polynucleotide ofclaim
 59. 66. The vector of claim 65, which is a plasmid.
 67. A hostcell comprising the vector of claim
 66. 68. The host cell of claim 67,which is a bacterium, an insect cell, a mammalian cell, or a plant cell.69. The host cell of claim 67, wherein the bacterium is Escherichiacoli.
 70. A method of producing a an attenuated Staphylococcusaureus-derived superantigen (SAg) SEA toxoid, comprising culturing thehost cell of claim 67, and recovering the oligopeptide.
 71. An isolatedpolynucleotide comprising a nucleic acid that encodes a multivalentoligopeptide comprising a fusion of two or more attenuatedStaphylococcus aureus-derived superantigen (SAg) toxoids arranged in anyorder, wherein the SAg toxoids can be the same or different, and whereinat least one of the SAg toxoids is the SEA toxoid of claim
 59. 72. Theisolated polynucleotide of claim 71 comprising the nucleotide sequenceSEQ ID NO:
 8. 73. A vector comprising the isolated polynucleotide ofclaim
 71. 74. The vector of claim 73, which is a plasmid.
 75. A hostcell comprising the vector of claim
 73. 76. The host cell of claim 75,which is a bacterium, an insect cell, a mammalian cell, or a plant cell.77. The host cell of claim 76, wherein the bacterium is Escherichiacoli.
 78. A method of producing a multivalent oligopeptide, the methodcomprising culturing the host cell of claim 75 and recovering theoligopeptide.